Choline binding proteins for anti-pneumococcal vaccines

ABSTRACT

The invention relates to bacterial choline binding proteins (CBPs) which bind choline. Such proteins are particularly desirable for vaccines against appropriate strains of Gram positive bacteria, particularly  streptococcus , and more particularly  pneumococcus . Also provided are DNA sequences encoding the bacterial choline binding proteins or fragment thereof, antibodies to the bacterial choline binding proteins, pharmaceutical compositions comprising the bacterial choline binding proteins, antibodies to the bacterial choline binding proteins suitable for use in passive immunization, and small molecule inhibitors of choline binding protein mediated adhesion. Methods for diagnosing the presence of the bacterial choline binding protein, or of the bacteria, are also provided. In a specific embodiment, a streptococcal choline binding protein is an enolase, which demonstrates strong affinity for fibronectin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation Application of U.S. Ser. No.09/829,382, filed Apr. 9, 2001, now U.S. Pat. No. 6,784,164, which is aDivisional of U.S. Ser. No. 08/847,065, filed May 1, 1997, now U.S. Pat.No. 6,245,335, which is a non-provisional application claiming priorityto Provisional Application Ser. No. 60/016,632, filed May 1, 1996.Applicants claim the benefit of these applications under 35 U.S.C. §120,and 35 U.S.C. §119(e), the contents all of which are incorporated hereinby reference in their entireties.

The present application claims priority to Provisional PatentApplication Ser. No. 60/016,632, filed May 1, 1996, pursuant to 35 USC119(e), the disclosure of which is incorporated herein by reference inits entirety.

The research leading to the present invention was supported, in part, byGrant Nos. AI 36445 and AI 38446 from the National Institutes of Health.Accordingly, the Government may have certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to choline binding proteins,methods for isolating choline binding proteins, and the genes encodingsuch proteins. The invention also relates to acellular vaccines toprovide protection from bacterial infection using such proteins, and toantibodies against such proteins for use in diagnosis and passive immunetherapy. In particular, the choline binding proteins of the inventionare useful as vaccines against pneumococcus. Where a choline bindingprotein demonstrates activity as an adhesion protein, it is also usefulas a competitive inhibitor of bacterial adhesion, or to discover smallmolecule antagonists of adhesion.

BACKGROUND OF THE INVENTION Antibacterial Vaccine Strategies

Exported proteins in bacteria participate in many diverse and essentialcell functions such as motility, signal transduction, macromoleculartransport and assembly, and the acquisition of essential nutrients. Forpathogenic bacteria, many exported proteins are virulence determinantsthat function as adhesions to colonize and thus infect the host or astoxins to protect the bacteria against the host's immune system[International Patent Publication No. WO 95/06732, published Mar. 9,1995 by Masure et al., which is specifically incorporated herein byreference in its entirety, for a review, see Hoepelman and Tuomanen,Infect. Immun., 60:1729-33 (1992)]. However, other exported proteins maynot directly mediate adhesion.

Since the development of the smallpox vaccine by Jenner in the 18thcentury, vaccination has been an important armament in the arsenalagainst infectious microorganisms. Prior to the introduction ofantibiotics, vaccination was the major hope for protecting populationsagainst viral or bacterial infection. With the advent of antibiotics inthe early 20th century, vaccination against bacterial infections becamemuch less important. However, the recent insurgence ofantibiotic-resistant strains of infectious bacteria has resulted in thereestablishment of the importance of anti-bacterial vaccines.

One possibility for an anti-bacterial vaccine is the use of killed orattenuated bacteria. However, there are several disadvantages of wholebacterial vaccines, including the possibility of a reversion of killedor attenuated bacteria to virulence due to incomplete killing orattenuation and the inclusion of toxic components as contaminants.

Another vaccine alternative is to immunize with the bacterialcarbohydrate capsule. Presently, vaccines against Streptococcuspneumoniae employ conjugates composed of the capsules of the 23 mostcommon serotypes of this bacterium. These vaccines are ineffective inindividuals most susceptible to pathological infection—the young, theold, and the immune compromised—because of its inability to elicit a Tcell immune response. A recent study has shown that this vaccine is only50% protective for these individuals [Shapiro et al., N. Engl. J. Med.325:1453-60 (1991)].

An alternative to whole bacterial vaccines are acellular vaccines orsubunit vaccines in which the antigen includes a bacterial surfaceprotein. These vaccines could potentially overcome the deficiencies ofwhole bacterial or capsule-based vaccines.

Moreover, given the importance of exported proteins to bacterialvirulence, these proteins are an important target for therapeuticintervention. Of particular importance are proteins that represent acommon antigen of all strains of a particular species of bacteria foruse in a vaccine that would protect against all strains of the bacteria.However, to date only a small number of exported proteins of Grampositive bacteria have been identified, and none of these represent acommon antigen for a particular species of bacteria.

Recently, apparent fusion proteins containing PhoA were exported inspecies of Gram positive and Gram negative bacteria (Pearce and Masure,1992, Abstr. Gen. Meet. Am. Soc. Microbiol. 92:127,abstract D-188). Thisabstract reports insertion of pneumococcal DNA upstream from the E. coliphoA gene lacking its signal sequence and promoter in a shuttle vectorcapable of expression in both E. coli and S. pneumoniae, and suggeststhat similar pathways for the translocation of exported proteins acrossthe plasma membranes must be found for both species of bacteria.

In previous studies, use of random translational gene fusions (PhoAmutagenesis) to identify and alter exported proteins in Streptococcuspneumoniae provided insight into putative exported proteins [Pearce etal., Mol. Microbiol., 9:1037 (1993); International Patent PublicationNo. WO 95/06732, published Mar. 9, 1995; U.S. patent application Ser.No. 08/116,541, filed Sep. 1, 1993; U.S. patent application Ser. No.08/245,511, filed May 18, 1994]. Coupling this gene fusion technology tobioactivity assays for pneumococcal adherence, the primary goal was togenetically identify and characterize immunogenic adhesion virulencedeterminants to eucaryotic cells that define the bacteria-hostrelationship and thus serve as vaccine candidates. Over 25 loci thateffect adherence have been identified as determinants of virulence.

In addition, the molecular mechanism of pathogenesis caused bypneumococcus are beginning to be defined [Cundell, et al., Infect.Immun. 63:2493-2498 (1995); Wizemann, et al., Proc. Natl. Acad. Sci. USA(1996); Cundell, et al., J. Cell Biol. S18A:45 (1994); Spellerberg, etal., Mol. Microb. (1996)]. The results of these efforts shows that manybacterial components participate in a complex coordinated process tocause disease. However, it is also apparent that this strategy hasproduced only a few potential vaccine candidates.

Of note in the search for exported pneumococcal proteins that might beattractive targets for a vaccine is pneumococcal surface protein A(PspA) [see Yother et al., J. Bacteriol., 174:610 (1992)]. PspA has beenreported to be a candidate for a S. pneumoniae vaccine as it has beenfound in all pneumococci to date; the purified protein can be used toelicit protective immunity in mice; and antibodies against the proteinconfer passive immunity in mice [Talkington et al., Microb. Pathog.13:343-355 (1992)]. However, PspA demonstrates antigenic variabilitybetween strains in the N-terminal half of the protein, which containsthe immunogenic and protection eliciting epitopes (Yother et al.,supra). This protein does not represent a common antigen for all strainsof S. pneumoniae, and therefore is not an optimal vaccine candidate.

Pneumococcal Choline Binding Proteins

Previous studies have shown that PspA, as well as one other surfaceexposed protein, LytA, the autolytic amidase, bind to teichoic acid(TA), an integral part of the cell wall of Streptococcus pneumoniae in acholine-dependent manner. TA contains a unique terminalphosphorylcholine moiety. PspA, a protein having a molecular weight of84 kDa, and which is highly variable, is released from the cell surfacewith high choline concentration. Lyt, or autolysin, is a 36 kDa protein,which lyses the pneumococcal cell wall (self lysis), but is not releasedfrom the cell by growth in high concentrations of choline, by washing in10% choline, or by growth in ethanolamine. Reports on choline bindingproteins include those by Sanchez-Puelles et al Gene 89:69-75 (1990),Briese and Hakenback Eur. J. Biochem. 146:417-427, Yother and White J.of Bacteriol. 176:2976-2985, Sanchez-Beato et al J. of Bacteriol.177:1098-1103, and Wren Micro. Review Mol. Microbiol. 5:797-803 (1991),which are hereby incorporated by reference in their entirety.

A variety of covalent and non-covalent mechanisms of attachment havebeen described for proteins decorating the surfaces of gram positivebacteria. Some streptococci and Clostridium sp. have phosphorylcholineas a unique component of the cell wall. This molecule is the terminalconstituent of the teichoic acid (C polysaccharide) and lipoteichoicacid (LTA) attached to the cell wall and plasma membrane of thesebacteria. A family of choline binding proteins (CPBs) have also beendescribed which serve a variety of cellular functions. These proteinsconsist of an N-terminal activity domain and a repeated C-terminalsignature choline binding domain that anchors these molecules to thesurface of the bacteria. This motif has been identified in theC-terminal regions of a secreted glycoprotein from Clostridiumacetobutylicum NCIB 88052 [Sanchez-Beato, et al., J. Bacteriol.177:1098-1103 (1995)], toxins A and B from Clostridium difficile [VonEichel-Streiber and Sauerborn, Gene 96:107-13 (1990); VonEichel-Streiber et al., J. Bacteriol. 174:6707-6710(1992)], aglucan-binding protein from Streptococcus mutans, severalglycosyltransferases from Streptococcus downei and S. mutans, the mureinhydrolase (LytA) from pneumococcus and pneumococcal lytic phage [Rondaet al., Eur. J. Biochem. 164:621-4,(1987); Diaz et al., J. Bacteriol.174:5516-25 (1992); Romero et al., Microb. Lett. 108:87-92 (1993);Yother and White, J. Bacteriol. 176:2976-85 (1994)], and a surfaceantigen (PspA) also from pneumococcus.

Pathology of Pneumococcal Adherence

S. pneumoniae is a gram positive bacteria which is a major cause ofinvasive infections such as sepsis and meningitis [Tuomanen et al., N.Engl. J. Med. 322:1280-1284(1995)]. The pneumococcus colonizes thenasopharyngeal epithelium and then penetrates the epithelium of the lungor nasopharynx in order to reach the vascular compartment. Suchtranslocation would involve, of necessity, passage from an epithelialsite through the underlying basement membrane/extracellular matrix andacross endothelia. Pneumococci have been demonstrated to adhere toepithelia, endothelia and basement membrane in vitro and in vivo[Plotkowski et al., Am. Rev. Respir. Dis., 134 (1986); Cundell andTuomanen, Microb Path., 17:361-374 (1994); Cundel et al., Nature,377:435-438 (1995); van der Flier et al., Infect. Immun., 63:4317-4322(1995).

Fibronectin is a mammalian glycoprotein present as a soluble dimer(molecular weight of 550 kDa) in body fluids such as plasma (200-700mg/ml), cerebrospinal fluid and amniotic fluid and as a less solublemultimer in the extracellular matrix and basement membrane [Ruoslahti,Ann. Rev. Biochem., 57:375-413 (1988)]. Fibronectin has specific bindingsites for a number of proteins including collagen, integrins, and twobinding sites for heparin. Many microorganisms bind fibronectin,including oral streptococci and some gram negative bacteria [Westerlundand Korhonen, Mol. Microbiol., 9:687-694 (1993)]. These diversepathogens all target the Type 1 repeats of the N-terminal heparinbinding domain of fibronectin. The cognate fibronectin binding proteinsdemonstrate a similar amino acid sequence motif consistent with bindingto the same target within fibronectin [Westerlund and Korhonen, 1993,supra]. In contrast to this pattern, Streptococcus pneumoniae was foundto adhere avidly to immobilized fibronectin at the carboxyterminalheparin binding domain [van der Flier et al., Infect. Immun.63:4317-4322, (1995)]. This domain of fibronection has a number ofbiological activities. It contains the major proteoglycan binding domain(Hep II) and also supports binding of the leukocyte integrin VLA-4 attwo regions in the type III connecting segment (IIICS) [Wayner et al.,Cell. Biol., 109:1321-1330 (1989; Guan and Hynes, Cell, 60:53-61 (1990);Mould et al., J. Biol. Chem., 266:3579-3585 (1991)]. The VLA-4 bindingdomain is distinct from that containing the RGD motif for bindingfibronectin by VLA-5 [Pierschbacher et al., Cell, 26:259-267 (1981).

The IIICS segment is subject to alternative splicing and is absent insome soluble forms of fibronectin [Guan and Hynes, 1990, supra].

VLA-4, α4β1 (CD49d/CD29), is an integrin present on lymphocytes,monocytes, muscle cells and melanoma cells which mediates binding toVCAM-1 on endothelial cells and myoblasts and to the IIICS domain offibronectin in the subendothelial matrix [Osborn et al., Cell,59:1203-1211 (1989); Mould et al., J. Biol. Chem. 254:4020-4024 (1990);Shimizu et al., Immunological Reviews, 114:109-143 (1990); Rosen et al.,Cell, 69:1107-1119 (1992)]. These interactions are important duringinfiltration of mononuclear cells to sites of inflammation, metastasisof melanoma cells and in myogenesis [McCarthy et al., J. Cell. Biol.102:179-188 (1986); Osborn et al., 1898, supra; Shimuzu et al., 1990,supra; Rosen et al., 1992, supra]. VLA-4 targets a 25 amino acid region(CS1) with the IIICS domain of fibronectin, an interaction which can beblocked by the tripeptide Leu-Asp-Val [Guan and Hynes, 1990, supra;Komoriya et al., J. Biol. Chem. 266:15075-15079 (1991); Mould et al.,1991, supra]. An homologous motif IDSP is present in the VLA4 bindingsites in VCAM-1 domains I and IV [Clements et al., J. Cell. Sci.,107:2127-2135 (1994)]. The binding sites on VLA-4 for VCAM-1 andfibronection have been suggested to be distinct but overlapping [Eliceset al., Cell, 50:577-584 (1990); Pulido et al., J. Biol. Chem.,266:10241-10245 (1991); Vonderheide and Springer, J. Exp. Med.,175:1433-1442 (1992); Makarem, J. Biol. Chem. 269:4005-4011 (1994)]. Theability of pneumococci to target the HepII region of fibronectin raisedthe possibility that these bacteria recognized the region common betweenHepII and VCAM-1 and that the binding was mediated by a bacterialversion of VLA-4. Such interactions could promote passage of pneumococciacross the basement membrane. The importance of VLA-4-VCAM-1interactions for leukocyte trafficking to brain also suggested a rolefor pneumococcal transmigration into the central nervous system inmeningitis.

Therefore, in view of the aforementioned deficiencies attendant withprior art methods of vaccinating against bacterial pathogens, it shouldbe apparent that there still exists a need in the art for identifyingprotein antigens suitable for use as subunit vaccines, and for use ininducing antibodies suitable for use in passive immunization.

The citation of any reference herein should not be construed as anadmission that such reference is available as prior art to theinvention.

SUMMARY OF THE INVENTION

In accordance with the present invention, bacterial surface antigens areprovided which are suitable for use in immunizing animals againstbacterial infection. More particularly, novel choline binding proteinsfrom streptococci, preferably pneumococci, are provided.

In a further embodiment, a method is provided for isolating andidentifying choline binding proteins, and the genes encoding them.

In its broadest aspect, the present invention extends to streptococcalsurface antigens, generally referred to herein as choline bindingproteins, having the following characteristics:

-   -   a) binding to choline; and    -   b) eluting from a choline affinity chromatographic column in the        presence of 10%, preferably at least 10%, choline in Dubelcco's        phosphate buffered saline (DPBS);        with the proviso that the bacterial surface antigen of the        present invention is not PspA or autolysin (LytA). In a        preferred aspect, the choline binding protein of the invention        has one or more of a characteristic selected from the group        consisting of:    -   c) inhibiting adherence of the bacteria to host cells;    -   d) being reactive with sera from patients infected or recovering        from infection with the bacteria;    -   e) being reactive with rabbit antisera generated against        purified choline binding proteins isolated from a choline        affinity column by elution in 10% choline, DPBS; and    -   f) labeled by fluorescein isothiocyanate (FITC) without        requiring bacterial lysis (i.e., in intact bacteria).

In a specific example, the bacterial surface antigen is isolated frompneumococcus.

In a still further aspect, the present invention extends to vaccinesbased on the choline binding proteins.

In a particular embodiment, the present invention relates to all membersof the herein disclosed family of bacterial surface antigens which bindcholine, with the proviso that this group does not include PspA or LytA.

In a preferred embodiment, the invention provides a choline bindingprotein with a high degree of sequence similarity to enolase,particularly B. subtilis enolase.

The present invention also relates to an isolated nucleic acids, such asrecombinant DNA molecule or cloned gene, or a degenerate variantthereof, which encodes a bacterial choline binding protein (CBP) of theinvention. Preferably, the nucleic acid molecule, in particular arecombinant DNA molecule or cloned gene, encoding the CBP has anucleotide sequence or is complementary to a DNA sequence or fragmentthereof which codes on expression for an amino acid having a sequence asfollows:

CBP112 (SEQ ID NO: 1) XENEGSTQAATSSNMAKTEHRKAAKQVVDE CBP90 (SEQ ID NO:2) AREFSLEKTR CBP84 (SEQ ID NO: 3) XREFSLEKTRNIGIMAHVDAGKT CBP80 (SEQ IDNO: 4) XKXXWQXKQYLKEDGSQAANEXVFDTA CBP78 (SEQ ID NO: 5)QKIIGIDLGTTNSAVAVLEGTESKIIANPE CBP70 (SEQ ID NO: 6) XXXEVAKXSQDTTTASCBP60 (SEQ ID NO: 7) XNERVKIVATLGPAVEGRG CBP50 (SEQ ID NO: 8)XIIXXVYAREVLDSRGNP CBP112-Int1 (SEQ ID NO: 9) EDRRNYHPTNTYK CBP112-Int2(SEQ ID NO: 10) XDDQQAEEDYA

-   Almost Full Length CBP50 (SEQ ID NO:19)-   Partial CBP112 (SEQ ID NO:21)

In a specific embodiment a nucleic acid of the invention, encodes atleast a portion (an internal fragment) of the choline binding proteinCBP50, preferably having a nucleotide sequence depicted in SEQ ID NO:18;or a portion of the 5′ region of the gene encoding binding proteinCBP112, preferably having a nucleotide sequence depicted in SEQ IDNO:20.

The DNA sequences encoding the CBPs of the present invention, orportions thereof, may be prepared as probes to screen for complementarysequences and genomic clones in the same or alternate species. Thepresent invention extends to probes so prepared that may be provided forscreening. For example, the probes may be prepared with a variety ofknown vectors, such as the phage λ vector. Such probes are useful fordiagnosis, e.g., to confirm a species or strain of Gram positivebacterial infection, as well as for cloning cDNA or genes encoding CBPs.

The present invention also includes CBPs having the activities notedherein, and that display the amino acid sequences set forth anddescribed above and selected from SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 19, and 21.

In a further embodiment of the invention, the full DNA sequence of therecombinant DNA molecule or cloned gene so determined may be operativelylinked to an expression control sequence which may be introduced into anappropriate host. The invention accordingly extends to unicellular hoststransformed with the cloned gene or recombinant DNA molecule comprisinga DNA sequence encoding the present CBP(s), and more particularly, theDNA sequences or fragments thereof determined from the sequences setforth above and in SEQ ID NOS:18 and 20.

According to other preferred features of certain preferred embodimentsof the present invention, a recombinant expression system is provided toproduce biologically active CBPs or immunologically reactive portionsthereof.

The concept of the bacterial surface antigens contemplates that specificfactors exist for correspondingly specific binding proteins, such asCBPs and the like, as described earlier. Accordingly, the exactstructure of each CBP will understandably vary so as to achieve thischoline-binding and activity specificity. It is this specificity and thedirect involvement of the CBP in the adherence of the bacteria, thatoffers the promise of a broad spectrum of diagnostic and therapeuticutilities.

The present invention naturally contemplates several means forpreparation of the CBPs, including as illustrated herein knownrecombinant techniques, and the invention is accordingly intended tocover such synthetic preparations within its scope. The isolation of theDNA and amino acid sequences disclosed herein facilitates thereproduction of the CBPs by such recombinant techniques, andaccordingly, the invention extends to expression vectors prepared fromthe disclosed DNA sequences for expression in host systems byrecombinant DNA techniques, and to the resulting transformed hosts.

A particular advantage of the present invention is that it provides forpreparation of sufficient quantities of CBPs for commercialization ofanti-CBP vaccines. A further advantage is that the invention providesfor preparation of multi-component vaccines containing two or more CBPs,thus broadening and increasing the potential effectiveness of thevaccine. In its primary aspect, the invention contemplates utilizing theCBPs of the invention, either separately or in combinations of two ormore, in vaccines for protection against pneumococcal infection.Preferably, a vaccine comprising two or more CBPs further comprisesPspA, LytA, or both. According to the invention, CBPs may be preparedrecombinantly. Alternatively, CBPs may be obtained from bacterialcultures, e.g., using the purification methods described and exemplifiedherein. In a specific embodiment, a mixture of CBPs from pneumococcusare obtained, e.g., by choline affinity chromatography, and are useddirectly without further purification to immunize an animal and elicitprotective antibodies. Such a mixture of choline affinity purifiedproteins may be obtained from bacteria that express PspA or that lackPspA expression (i.e., PspA⁻ bacteria as exemplified herein).

In another aspect, the genes (e.g., cDNA) encoding one or more CBPs ofthe invention are engineered in a transgenic vector for expression in amammalia host in vitro, as a nucleic acid-based vaccine.

In yet another embodiment, the CBPs of the invention are used togenerate antibodies for passive immunization, diagnostics, or screening.In a specific example, infra, passive immunization prevents death frompneumococcal infection in a murine model.

The diagnostic utility of the present invention extends to the use ofbinding partners, notably antibodies, to the present CBPs in assays toscreen for bacterial infection.

Antibodies against the CBP(s) include naturally raised and recombinantlyprepared antibodies. Such antibodies could be used to screen expressionlibraries to obtain the gene or genes that encode the CBP(s). These mayinclude both polyclonal and monoclonal antibodies prepared by knowngenetic techniques, as well as bi-specific (chimeric) antibodies, andantibodies including other functionalities suiting them for additionaldiagnostic use conjunctive with their capability of modulating bacterialadherence.

Thus, the CBPs, their analogs and/or analogs, and antibodies that may beraised thereto, are capable of use in connection with various diagnostictechniques, including immunoassays, such as a radioimmunoassay, usingfor example, an antibody to the CBP that has been labeled by eitherradioactive addition, or radioiodination.

In an immunoassay, a control quantity of the antagonists or antibodiesthereto, or the like may be prepared and labeled with an enzyme, aspecific binding partner and/or a radioactive element, and may then beintroduced into a cellular sample. After the labeled material or itsbinding partner(s) has had an opportunity to react with sites within thesample, the resulting mass may be examined by known techniques, whichmay vary with the nature of the label attached.

In the instance where a radioactive label, such as the isotopes ³H, ¹⁴C,³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I, and ⁸⁶Re areused, known currently available counting procedures may be utilized. Inthe instance where the label is an enzyme, detection may be accomplishedby any of the presently utilized colorimetric, spectrophotometric,fluorospectrophotometric, amperometric or gasometric techniques known inthe art.

The present invention includes an assay system which may be prepared inthe form of a test kit for the quantitative analysis of the extent ofthe presence of the bacterial infection, or to identify drugs or otheragents that may mimic or block such infection. The system or test kitmay comprise a labeled component prepared by one of the radioactiveand/or enzymatic techniques discussed herein, coupling a label to theCBPs, their agonists and/or antagonists, and one or more additionalimmunochemical reagents, at least one of which is a free or immobilizedligand, capable either of binding with the labeled component, itsbinding partner, one of the components to be determined or their bindingpartner(s).

In particular, the proteins of CBPs whose sequences are presented in SEQID, NOS:1-10, 19 and 20 herein, their antibodies, agonists, antagonists,or active fragments thereof, could be prepared in pharmaceuticalformulations for administration in instances wherein antibiotic therapyis appropriate, such as to treat or prevent bacterial infection. Suchfree proteins could compete with bacterial CBP function, thusinterfering with bacterial pathological activity such as adhesion.

In a preferred aspect, the invention provides a method and associatedcompositions for treating infection with a bacterium that expresses astreptococcal choline binding protein comprising administeringpulmonarily an adhesion inhibitory agent selected from the groupconsisting of a choline binding protein having the followingcharacteristics:

-   -   choline-binding activity; and    -   elution from a chromatographic column in the presence of 10%        choline;        with the proviso that the streptococcal choline binding protein        is not PspA or autolysin (LytA), an antibody to the choline        binding protein, an enolase, a hindered cationic small molecule,        the peptide WQPPRARI (SEQ ID NO:11), and an antibody specific        for an epitope having the amino acid sequence WQPPRARI (SEQ ID        NO:11). Preferably, the hindered cationic small molecule is        selected from the group consisting of lysine, choline, and        arginine. In a further embodiment, the adhesion inhibitory agent        is administered with another drug, such as an antibiotic, a        steroid, a non-steroidal anti-inflammatory drug, etc.

Accordingly, it is a principal object of the present invention toprovide a CBP and its subunits in purified form.

It is a further object of the present invention to provide antibodies tothe CBP and its subunits, and methods for their preparation, includingrecombinant means.

It is a further object of the present invention to provide a method fordetecting the presence of the CBP and its subunits in mammals in whichinvasive, spontaneous, or idiopathic pathological states are suspectedto be present.

It is a still further object of the present invention to provide methodfor the treatment of mammals to control the amount or activity of thebacteria, the CBP or subunits thereof, so as to alter the adverseconsequences of such presence or activity, or where beneficial, toenhance such activity.

It is a still further object of the present invention to provide amethod for the treatment of mammals to control the amount or activity ofthe bacteria or its subunits, so as to treat or avert the adverseconsequences of invasive, spontaneous, or idiopathic pathologicalstates.

It is a still further object of the present invention to providepharmaceutical compositions for use in therapeutic methods whichcomprise or are based upon the CBP, its subunits, or their bindingpartner(s).

Other objects and advantages will become apparent to those skilled inthe art from a review of the ensuing description which proceeds withreference to the following illustrative drawings and DetailedDescription of the Invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Western blot of a 6% SDS-PAGE on which bacterial surfaceproteins were separated. Lane A represents FITC-labelled surfaceproteins from LM91 (PspA⁻). Lane B represents FITC-labelled surfaceproteins from LM91 after choline affinity chromatography. Lane Crepresents LM91 proteins after choline affinity chromatography (CBRO).“FITC” indicates anti-FITC (mouse) antibody was used. “Convalesc.”indicates human convalescent (post-pneumococcal infection) antibody wasused. “CBP” indicates anti-CBP antibody (rabbit) was used.

FIG. 2. Western blot of a 10% SDS-PAGE as described in FIG. 1.

FIG. 3. Adhesion assay of PN R6 to lung cell A549.

FIG. 4. Adhesion assay of PN R6 to HUVEC (endothelial) cells.

FIG. 5. PRIOR ART. Schematic drawing of fibronectin and its fragments.

FIG. 6. Direct adherence of pneumococcus to recombinant fragments of the33 kDa heparin 11 binding domain.

FIG. 7. Direct adherence of pneumococcus to synthetic peptides based onthe heparin 11 type III #14 and IIICS regions.

FIG. 8. Inhibition of S. pneumiae adherence by choline. Bacteria werepreincubated with choline, eluted proteins and choline were washed away,and bacterial adhesion to fibronectin evaluated.

FIG. 9. Inhibition of pneumococcal adherence to fibronectin by ananti-choline antibody, TEPC15.

FIG. 10. Comparison of amino terminal sequence of 50 kDa protein with B.subtilis enolase.

FIG. 11. Inhibition of pneumococcal adherence to fibronectin by yeastenolase (Sigma).

FIG. 12. Inhibition of pneumococcal adherence to fibronectin byL-lysine.

FIG. 13. SDS-PAGE of eluate from a fibronectin-coupled CnBr-activatedSEPHAROSE 4B column (lane A), and yeast enolase (Sigma) (lane. B).French Press lysate of S. pneumoniae was applied to thefibronectin-SEPHAROSE column. The column was washed and adsorbedproteins eluted in 0.01, 0.1, and 0.5 M L-lysine. The 0.5 M lysineeluate was analyzed by SDS-PAGE with silver stain. The eluted proteinhad an apparent molecular weight comparable to yeast enolase.

FIG. 14. DNA Sequence comparison of DNA for the 50 kDa protein (SEQ IDNO:14) and B. subtilis enolase (SEQ ID NO:15). The sequences were 74%identical.

FIG. 15. Deduced amino acid sequence comparison for the 50 kDa protein(SEQ ID NO:16) and B. subtilis enolase (SEQ ID NO:17). These sequenceswere 72% identical, with 85% positives.

FIG. 16 Passive protection of mice against sepsis with anti-CBPantiserum. (A) CF1 mice challenged intraperitoneally with D39 (type 2)pneumococci at a dose of either 4.5×10⁴ (n=10 per group; ●) or 8.4×10⁴(n=5 per group; ▪) in 0.5 ml PBS. Experimental groups (-) were treatedwith 0.5 ml of 1:10 diluted rabbit anti-CBP serum one hour afterinoculation with bacteria. Control mice (--) received pre-immune serum.Percent survival was evaluated daily over a six day period. (B) Passiveprotection against challenge with heterologous serotype SIII (type 3;n=5 per group; □) was performed as in A with a dose of 200 cfu. Bacteriawere preincubated with antiserum (●) for 30 min prior to intraperitonealinoculation. Blood was sampled at 24 hours for cfu and survival wasassessed every 12 hours for 72 hours.

FIG. 17. Competitive inhibition of pneumococcal adherence to human cellsby exogenous purified CBPs. (A) Type II lung cells (LC) or (B)endothelial cells (EC) were preincubated for 15 min with CBP preparation(1 mg/ml), washed and then incubated with FITC-labelled R6 pneumococcus.E. coli DH5a was used as a control. Adherence was quantitated byfluorescence intensity. (C) Dose dependence of the ability of CBPs toinhibit adherence of FITC-labelled pneumococci.

FIG. 18. Adherence of CbpA⁻ mutant to human cells and glycoconjugates.(A) Monolayers of Type II lung cell line A549 (LC) or (B) umbilical veinendothelial cells (EC) were activated by treatment with TNFα (10 ng/mlfor 2 h) or IL-1 (5 ng/ml for 4 h) or were untreated (resting). R6 andthe CbpA⁻ mutant were fluorescein labelled and overlaid onto themonolayers for 30 min. Nonadherent bacteria were washed away andadherence was quantitated visually and expressed as a % of control, i.e.100%=498±81 or 174±81 R6 bacteria per 100 resting LC and ECrespectively. The experiment was performed 5 times with 6-8 wells percondition. (C) Terasaki plates were coated with 6′ sialyllactose (6′SL),lacto-N-neotetraose (LnNT), N-acetylgalactosamine-β1,4-galactose HSA,(GlcNAc-β1,4-Gal), N-acetylgalactosamine-β1,3-galactose HSA(GlcNAcβ1,3-Gal), or HSA at 100 μM. R6 (solid bars) or CbpA⁻ (stippledbars) were grown to an OD₆₂₀ of 0.4 for all experiments except thosewith 6′SL (OD620 of 0.6). Fluorescein labelled bacteria were placed inthe wells for 30 min, the wells were washed, and adherent bacteria werecounted visually per 40× field. Values are expressed as a % of control,i.e. 100%=133±24 R6 per 40 × field of 6′SL well, 32±8, 20±, and 15±4 for6′SL, LnNT, GlcNAc-β1,4-gal, and GlcNAcβ1,3-Gal, respectively. Theexperiment was performed 3 times with 6-12 wells per condition.

FIG. 19. The contribution of CbpA to pneumococcal carriage in infantrats. The ability to colonize the nasopharynx of 5 day old rats werecompared between the control strains D39 (hatched), D39 (iga⁻)(stippled) and an isogenic cbpA deficient strain (solid). The ordinateindicates the time following inoculation and the abscissa the number ofcolonies in nasal washes (mean±SD, n=20 per group).

DETAILED DESCRIPTION

As noted above, the present invention is directed to bacterial surfaceantigens that are suitable for use in immunizing animals againstbacterial infection. More particularly, novel choline binding proteinsfrom pneumococci are provided. These proteins, which are found at thesurface of pneumococci, when formulated with an appropriate adjuvant,are used in vaccines for protection against pneumococci, and againstother bacteria with cross-reactive choline binding proteins.

As has been previously reported, S. pneumoniae adheres to fibronectin ata site within the carboxy-terminal heparin II binding domain [Flier etal., Infect. Immun. 63:4317 (1995)]. An eight amino acid stretch withinthe type III # 14 repeat supports adherence. The present invention isbased, in part, on the discovery that the pneumococcal adhesin forfibronectin appears to be a choline-binding protein of approximately 50kDa. This protein has significant homology to the glycolytic enzymeenolase. For example, preincubation of S. pneumoniae with rsVCAMinhibits adherence to whole fibronectin by 96%, and S. pneumoniae adheredirectly to rsVCAM with 6% of adherence to whole fibronectin. Inaddition, pneumococci bind directly to a synthetic peptide, Fn5, basedon the Heparin II type III #14 region, having the sequence WQPPRARI (SEQID NO:11). Antibody to Fn5 inhibits adherence of S. pneumoniae to wholefibronectin by greater than 70% (1:100). The data exemplified hereinshow that S. pneumoniae grown in ethanolamine vs. choline results in agreater than 90% decrease in adherence. Preparations of CBPcompetitively inhibit adherence of S. pneumoniae to fibronectin by75-90%. Anti-choline antibody inhibits adherence of S. pneumoniae tofibronectin by greater than 95% at dilution of 1:10 to 1:5000.Pretreatment of S. pneumoniae in 10% choline results in greater than 50%decrease in adherence to fibronectin.

In a particular embodiment, the present invention relates to all membersof the herein disclosed pneumococcal CBPs, with the proviso that thisgroup does not include Lyt or PspA. In a specific embodiment, a CBP ofthe invention is a homolog of enolase.

The possibilities both diagnostic and therapeutic that are raised by theexistence of the CBP, derive from the fact that the CBP appears toparticipate in direct and causal protein-protein interaction between thebacteria and its host cell. As suggested earlier and elaborated furtheron herein, the present invention contemplates pharmaceuticalintervention in the binding reaction in which the CBP is implicated, tomodulate the activity initiated by the binding of the CBP.

In a further embodiment, a method is provided for isolating andidentifying choline binding proteins, and the genes encoding them. Moreparticularly, isolation of the genes encoding choline binding proteinsof the invention allows for recombinant production of the proteins,which greatly increases the ability to generate cost-effective vaccines.

The choline binding protein streptococcal surface antigens of theinvention have the following characteristics:

-   -   a) binding to choline; and    -   b) eluting from a choline affinity chromatographic column in the        presence of 10% choline in Dulbecco's phosphate buffered saline        (DPBS);        with the proviso that the streptococcal surface antigen of the        present invention is not PspA or autolysin (LytA). In a        preferred aspect, the choline binding protein of the invention        has one or more of a characteristic selected from the group        consisting of:    -   c) inhibiting adherence of the bacteria to host cells;    -   d) being reactive with sera from patients infected or recovering        from infection with the bacteria;    -   e) being reactive with rabbit antisera generated against        purified choline binding proteins isolated from a choline        affinity column by elution in 10% choline, DPBS; and    -   f) labeled by fluorescein isothiocyanate (FITC) without        requiring bacterial lysis (i.e., in intact bacteria).

In a specific example, infra, the streptococcal surface antigen isisolated from pneumococcus by affinity chromatography on a cholineagarose column by elution with 10% choline.

In a further embodiment, exemplified herein, a peptide based on theheparin II type III #14 region of fibronectin binds the streptococcalenolase homolog of the invention. In a specific embodiment, the peptideis Fn5 having an amino acid sequence WQPPRARI (SEQ ID NO:11). Wholepneumococci adhere to this peptide prepared synthetically. Thus, thefree peptide would be expected to inhibit enolase-mediated adherence tofibronectin in vivo. In addition, an antibody specific for this peptideinhibits pneumococcal adherence to fibronectin. In a specificembodiment, an anti Fn5 antibody inhibits adherence of S. pneumoniae towhole fibronectin by greater than 70%.

The term “bacterial” used herein refers to Gram positive bacteria withcholine binding proteins homologous to the proteins exemplified herein.The present invention is more particularly directed to streptococcalCBPs and most particularly to pneumococcal CBPs.

The terms “bacterial (or streptococcal or pneumococcal) surfaceantigen”, “choline binding protein (CBP)” and any variants notspecifically listed, may be used herein interchangeably, and as usedthroughout the present application and claims refer to proteinaceousmaterial including single or multiple proteins, and extends to thoseproteins having the amino acid sequence data described herein andidentified by (SEQ ID NOS:1-10, 19, and 20), and the profile ofactivities set forth herein and in the Claims. Accordingly, proteinsdisplaying substantially equivalent or altered activity are likewisecontemplated. These modifications may be deliberate, for example, suchas modifications obtained through site-directed mutagenesis, or may beaccidental, such as those obtained through mutations in hosts that areproducers of the complex or its named subunits. Also, the terms“bacterial (or streptococcal or pneumococcal) surface antigen”, and“choline binding protein (CBP)” are intended to include within theirscope proteins specifically recited herein as well as all substantiallyhomologous analogs and allelic variations.

The term “enolase” refers to the enzyme 2-phospho-D-glyceratehydrolyase.

The amino acid residues described herein are preferred to be in the “L”isomeric form. However, residues in the “D” isomeric form can besubstituted for any L-amino acid residue, as long as the desiredfunctional property of immunoglobulin-binding is retained by thepolypeptide. NH₂ refers to the free amino group present at the aminoterminus of a polypeptide. COOH refers to the free carboxy group presentat the carboxy terminus of a polypeptide. Abbreviations used herein arein keeping with standard polypeptide nomenclature, J. Biol. Chem.,243:3552-59 (1969).

It should be noted that all amino-acid residue sequences are representedherein by formulae whose left and right orientation is in theconventional direction of amino-terminus to carboxy-terminus.Furthermore, it should be noted that a dash at the beginning or end ofan amino acid residue sequence indicates a peptide bond to a furthersequence of one or more amino-acid residues.

Purification of CBPs

Teichoic acid (TA), an integral part of the cell wall of Streptococcuspneumoniae contains a unique terminal phosphorylcholine moiety. Cholineaffinity chromatography or Mono-Q Sepharose, a close relative of cholinewere used to purify the CBPs. It is important to note that initiallythese proteins were purified from a capsulated strain of pneumococcusthat was genetically altered not to produce PspA, a major CBP. Thepurification schemes are as follows:

-   -   Whole bacteria are incubated with either choline-agarose or        Mono-Q Sepharose.    -   Bacteria are lysed with detergent and unbound material washed        with 0.5 M NaCl.    -   CBPs are eluted with either with 0.5 M choline chloride or with        a linear choline chloride gradient. The molecular masses of        proteins purified by these two methods are summarized in Table 1        (see FIG. 1).

TABLE 1 Criteria for the identification of choline binding proteins asvaccine candidates from a capsulated PspA deficient strain ofpneumococcus* Labeling of surface Rabbit antisera to whole Humanconvalescent Purification Scheme SDS-PAGE 10% proteins bacteria serumCell lysate 112 kDa  90 kDa 200 kDa  112 kDa  200 kDa 130 kDa  (0.5%Triton × 100) 80 kDa 75 kDa 90 kDa 84 kDa 112 kDa  90 kDa 60 kDa 53 kDa75 kDa 53 kDa 84 kDa 82 kDa 37 kDa 30 kDa 48 kDa 45 kDa 80 kDa 75 kDa 28kDa 20 kDa 37 kDa 35 kDa 60 kDa 53 kDa Mono-Q Anion 5% choline gradient:112 kDa  84 kDa 53 kDa 37 kDa Choline-Agarose 200 kDa  112 kDa  112 kDa 95 kDa 75 kDa <35 kDa   112 kDa  (Elution with 0.5 M 90 kDa 84 kDa 80kDa 75 kDa 80 kDa choline chloride) 82 kDa 80 kDa 70 kDa 37 kDa 75 kDa75 kDa 60 kDa 40 kDa 37 kDa *Bold faced characters represent proteins ofinterest that are potential vaccine candidates.

Purification of CBPs from the choline agarose affinity chromatography ispreferred, with at least 9 proteins with molecular masses ranging from200 to 40 kDa identifiable this way. A band of 37 kDa was also detectedand shown to be LytA with antisera specific for this protein. This is awell characterized CBP and thus served as a positive control.

Criteria for Vaccine Candidates

CBPs may be subjected to a variety of tests to determine if they aregood vaccine candidates The criteria for vaccine development, and thecharacteristics of the isolated CBPs are summarized below.

The CBPs must be surface exposed. Whole bacteria may be chemicallylabeled with FITC (Fluorescein isothiocyanate) and labeled proteinsdetected with antisera specific for FITC. (Table 1 and FIG. 1). The CBPs112, 75, and 80, as well as LytA, were effectively labeled with FITCsuggesting that these proteins were surface exposed.

Vaccine candidates must be immunogenic therefore candidate CBPs shouldreact with human convalescent sera. The CBPs 112, 75, and 80 gave astrong signal with pooled human antisera obtained from individualsrecovering from pneumococcal disease (FIG. 1).

Vaccine candidates must be antigenic. The CBPs from choline agarosechromatography were injected into rabbits and sera tested for crossreactivity. Several proteins produced a strong signal. Prominent wereCBPs 112, 90, 84 and 70, and 50 (FIGS. 1 and 2).

A good vaccine candidate may block adherence to target cell receptorspresent within the host at the critical sites of infection. Compared tocontrols, the CBP fraction blocked pneumococcal adherence by 45% to LCand 89% to EC in a dose dependent manner (FIG. 3). Based on theseresults it is most likely that the fraction of CBPs contain adhesinsinvolved in the binding of bacteria to eucaryotic target cells. Thecontribution of each of these CBPs to block the adhesive properties ofparental bacteria can be assessed, for example, to block the binding ofpneumococcus to epithelial (type II lung cells), endothelial (humanumbilical vein endothelial cells) cells, and immobilized glycoconjugatesthat contain GlcNAcβ1-4Gal, GlcNAcβ1-3Gal, GlcNAc, or other sugars thathave been shown to be analogs for eucaryotic receptors.

It should be noted that not every CBP may function as an adhesin,similarly, adhesion activity may be a collateral characteristic of CBPs.

A preferred vaccine candidate will elicit a protective immune responsewithout antigenic variability among in clinical serotypes. Antisera(either monoclonal or polyclonal) to each CBP will be generated todetermine if the native and the recombinant CBPs are immunogenic. CBPspecific antibodies will be used to screen relevant pneumococcalserotypes for antigenic variability.

In a more preferred aspect, antibodies to the CBPs will be tested toconfirm that they protect against pneumococcal infection, preferably ofvarious strains or serotypes. For example, passively or activelyimmunized animals are challenged with pneumococcus in models forbacteremia or colonization, or preferably both.

Other criteria to consider in selection of a preferred CBP as a vaccinecandidate include testing CBP defective mutants for attenuation ofvirulence in animal models for bacteremia or colonization efficacy aloneor in combination or coupled to a capsular polysaccharide. For example,preliminary data show that CBP112 is expressed in virulent transparentbacteria, but that expression is diminished in avirulent opaquebacteria.

Genes Encoding CBPs

As stated above, the present invention also relates to a recombinant DNAmolecule or cloned gene, or a degenerate variant thereof, which encodesa CBP, or a fragment thereof, that possesses a molecular weight ofbetween about 50 kDa and 112 kDa, preferably which has an amino acidsequence set forth in (SEQ ID NOS:1-10, 19 and 20). In a specificaspect, a nucleic acid molecule, in particular a recombinant DNAmolecule or cloned gene, encoding the 50-112 kDa CBP has a nucleotidesequence or is complementary to a DNA sequence shown in (SEQ ID NO:18 or20).

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See; e.g., Sambrook et al, “Molecular Cloning:A Laboratory Manual” (1989); “Current Protocols in Molecular Biology”Volumes I-III [Ausubel, R. M., ed. (1994)]; “Cell Biology: A LaboratoryHandbook” Volumes I-IlI [J. E. Celis, ed. (1994))]; “Current Protocolsin Immunology” Volumes I-III [Coligan, J. E., ed. (1994)];“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “TranscriptionAnd Translation” [B. D. Hames & S. J. Higgins, eds. (1984)]; “AnimalCell Culture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells AndEnzymes” [IRL Press, (1986)]; B. Perbal, “A Practical Guide To MolecularCloning” (1984).

Therefore, if appearing herein, the following terms shall have thedefinitions set out below.

A “replicon” is any genetic element (e.g., plasmid, chromosome, virus)that functions as an autonomous unit of DNA replication in vivo; i.e.,capable of replication under its own control.

A “vector” is a replicon, such as plasmid, phage or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment.

A “DNA molecule” refers to the polymeric form of deoxyribonucleotides(adenine, guanine, thymine, or cytosine) in its either single strandedform, or a double-stranded helix. This term refers only to the primaryand secondary structure of the molecule, and does not limit it to anyparticular tertiary forms. Thus, this term includes double-stranded DNAfound, inter alia, in linear DNA molecules (e.g., restrictionfragments), viruses, plasmids, and chromosomes. In discussing thestructure of particular double-stranded DNA molecules, sequences may bedescribed herein according to the normal convention of giving only thesequence in the 5′ to 3′ direction along the nontranscribed strand ofDNA (i.e., the strand having a sequence homologous to the mRNA).

An “origin of replication” refers to those DNA sequences thatparticipate in DNA synthesis.

A DNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in vivo when placed underthe control of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxyl) terminus. Acoding sequence can include, but is not limited to, prokaryoticsequences, cDNA from eukaryotic mRNA, genomic DNA sequences fromeukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. Apolyadenylation signal and transcription termination sequence willusually be located 3′ to the coding sequence.

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, polyadenylation signals,terminators, and the like, that provide for the expression of a codingsequence in a host cell.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined by mapping with nuclease S1), as well as protein binding domains(consensus sequences) responsible for the binding of RNA polymerase.Eukaryotic promoters will often, but not always, contain “TATA” boxesand “CAT” boxes. Prokaryotic promoters contain Shine-Dalgarno sequencesin addition to the −10 and −35 consensus sequences.

An “expression control sequence” is a DNA sequence that controls andregulates the transcription and translation of another DNA sequence. Acoding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then translated intothe protein encoded by the coding sequence.

A “signal sequence” can be included before the coding sequence. Thissequence encodes a signal peptide, N-terminal to the polypeptide, thatcommunicates to the host cell to direct the polypeptide to the cellsurface or secrete the polypeptide into the media, and this signalpeptide is clipped off by the host cell before the protein leaves thecell. Signal sequences can be found associated with a variety ofproteins native to prokaryotes and eukaryotes.

The term “oligonucleotide,” as used herein in referring to the probe ofthe present invention, is defined as a molecule comprised of two or moreribonucleotides, preferably more than three. Its exact size will dependupon many factors which, in turn, depend upon the ultimate function anduse of the oligonucleotide.

The term “primer” as used herein refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product, which is complementary to a nucleic acid strand, isinduced, i.e., in the presence of nucleotides and an inducing agent suchas a DNA polymerase and at a suitable temperature and pH. The primer maybe either single-stranded or double-stranded and must be sufficientlylong to prime the synthesis of the desired extension product in thepresence of the inducing agent. The exact length of the primer willdepend upon many factors, including temperature, source of primer anduse of the method. For example, for diagnostic applications, dependingon the complexity of the target sequence, the oligonucleotide primertypically contains 15-25 or more nucleotides, although it may containfewer nucleotides.

A DNA sequence is “operatively linked” to an expression control sequencewhen the expression control sequence controls and regulates thetranscription and translation of that DNA sequence. The term“operatively linked” includes having an appropriate start signal (e.g.,ATG) in front of the DNA sequence to be expressed and maintaining thecorrect reading frame to permit expression of the DNA sequence under thecontrol of the expression control sequence and production of the desiredproduct encoded by the DNA sequence. If a gene that one desires toinsert into a recombinant DNA molecule does not contain an appropriatestart signal, such a start signal can be inserted in front of the gene.

The term “standard hybridization conditions” refers to salt andtemperature conditions substantially equivalent to 5×SSC and 65° C. forboth hybridization and wash. However, one skilled in the art willappreciate that such “standard hybridization conditions” are dependenton particular conditions including the concentration of sodium andmagnesium in the buffer, nucleotide sequence length and concentration,percent mismatch, percent formamide, and the like. Also important in thedetermination of “standard hybridization conditions” is whether the twosequences hybridizing are RNA-RNA, DNA-DNA or RNA-DNA. Such standardhybridization conditions are easily determined by one skilled in the artaccording to well known formulae, wherein hybridization is typically10-20° C. below the predicted or determined T_(m) with washes of higherstringency, if desired.

The primers herein are selected to be “substantially” complementary todifferent strands of a particular target DNA sequence. This means thatthe primers must be sufficiently complementary to hybridize with theirrespective strands. Therefore, the primer sequence need not reflect theexact sequence of the template. For example, a non-complementarynucleotide fragment may be attached to the 5′ end of the primer, withthe remainder of the primer sequence being complementary to the strand.Alternatively, non-complementary bases or longer sequences can beinterspersed into the primer, provided that the primer sequence hassufficient complementarity with the sequence of the strand to hybridizetherewith and thereby form the template for the synthesis of theextension product.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

A cell has been “transformed” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. The transforming DNA may or maynot be integrated (covalently linked) into chromosomal DNA making up thegenome of the cell. In prokaryotes, yeast, and mammalian cells forexample, the transforming DNA may be maintained on an episomal elementsuch as a plasmid. With respect to eukaryotic cells, a stablytransformed cell is one in which the transforming DNA has becomeintegrated into a chromosome so that it is inherited by daughter cellsthrough chromosome replication. This stability is demonstrated by theability of the eukaryotic cell to establish cell lines or clonescomprised of a population of daughter cells containing the transformingDNA. A “clone” is a population of cells derived from a single cell orcommon ancestor by mitosis. A “cell line” is a clone of a primary cellthat is capable of stable growth in vitro for many generations.

Two DNA sequences are “substantially homologous” when at least about 75%(preferably at least about 80%, and most preferably at least about 90 or95%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II,supra; Nucleic Acid Hybridization, supra.

It should be appreciated that also within the scope of the presentinvention are DNA sequences encoding CBPs which code for a CBP havingthe same amino acid sequence as one of SEQ ID NOS:1-5, but which aredegenerate to an oligonucleotide rep resented by the purified natural(i.e., native) gene, e.g., by SEQ ID NO:11. By “degenerate to” is meantthat a different three-letter codon is used to specify a particularamino acid. It is well known in the art that the following codons can beused interchangeably to code for each specific amino acid:

Phenylalanine (Phe or F) UUU or UUC Leucine (Leu or L) UUA or UUG or CUUor CUC or CUA or CUG Isoleucine (Ile or I) AUU or AUC or AUA Methionine(Met or M) AUG Valine (Val or V) GUU or GUC of GUA or GUG Serine (Ser orS) UCU or UCC or UCA or UCG or AGU or AGC Proline (Pro or P) CCU or CCCor CCA or CCG Threonine (Thr or T) ACU or ACC or ACA or ACG Alanine (Alaor A) GCU or GCG or GCA or GCC Tyrosine (Tyr or Y) UAU or UAC Histidine(His or H) CAU or CAC Glutamine (Gln or Q) CAA or CAG Asparagine (Asn orN) AAU or AAC Lysine (Lys or K) AAA or AAG Aspartic Acid (Asp or D) GAUor GAC Glutamic Acid (Glu or E) GAA or GAG Cysteine (Cys or C) UGU orUGC Arginine (Arg or R) CGU or CGC or CGA or CGG or AGA or AGG Glycine(Gly or G) GGU or GGC or GGA or GGG Termination codon UAA (ochre) or UAG(amber) or UGA (opal)

It should be understood that the codons specified above are for RNAsequences. The corresponding codons for DNA have a T substituted for U.

Completely degenerate oligonucleotides can be designed based on theamino acid sequence encoding the CBP, taking into account the abovedegenerates. Likewise, where a codon encoding an amino acid is not knownwith certainty, e.g., due to degeneracy of the genetic code, inosine maybe included at the unknown position.

Mutations can be made in a nucleic acid encoding a CBP such that aparticular codon is changed to a codon which codes for a different aminoacid. Such a mutation is generally made by making the fewest nucleotidechanges possible. A substitution mutation of this sort can be made tochange an amino acid in the resulting protein in a non-conservativemanner (i.e., by changing the codon from an amino acid belonging to agrouping of amino acids having a particular size or characteristic to anamino acid belonging to another grouping) or in a conservative manner(i.e., by changing the codon from an amino acid belonging to a groupingof amino acids having a particular size or characteristic to an aminoacid belonging to the same grouping). Such a conservative changegenerally leads to less change in the structure and function of theresulting protein. A non-conservative change is more likely to alter thestructure, activity or function of the resulting protein. The presentinvention should be considered to include sequences containingconservative changes which do not significantly alter the activity orbinding characteristics of the resulting protein. Substitutes for anamino acid within the sequence may be selected from other members of theclass to which the amino acid belongs. For example, the nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan and methionine. Amino acidscontaining aromatic ring structures are phenylalanine, tryptophan, andtyrosine. The polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine. The positivelycharged (basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid. Such alterations will not be expected to affect apparentmolecular weight as determined by polyacrylamide gel electrophoresis, orisoelectric point.

Particularly preferred substitutions are:

-   -   Lys for Arg and vice versa such that a positive charge may be        maintained;    -   Glu for Asp and vice versa such that a negative charge may be        maintained;    -   Ser for Thr such that a free —OH can be maintained; and    -   Gln for Asn such that a free NH₂ can be maintained.

Two amino acid sequences are “substantially homologous” when at leastabout 70% of the amino acid residues (preferably at least about 80%, andmost preferably at least about 90 or 95%) are identical, or representconservative substitutions.

A “heterologous” region of the DNA construct is an identifiable segmentof DNA within a larger DNA molecule that is not found in associationwith the larger molecule in nature. Thus, when the heterologous regionencodes a mammalian gene, the gene will usually be flanked by DNA thatdoes not flank the mammalian genomic DNA in the genome of the sourceorganism. Another example of a heterologous coding sequence is aconstruct where the coding sequence itself is not found in nature (e.g.,a cDNA where the genomic coding sequence contains introns, or syntheticsequences having codons different than the native gene). Allelicvariations or naturally-occurring mutational events do not give rise toa heterologous region of DNA as defined herein.

The present invention extends to the preparation of oligonucleotidesthat may be used to hybridize with nucleic acids encoding CBPs.

As mentioned above, a DNA sequence encoding a CBP can be preparedsynthetically rather than cloned. The DNA sequence can be designed withthe appropriate codons for the CBP amino acid sequence. In general, onewill select preferred codons for the intended host if th sequence willbe used for expression. The complete sequence is assembled fromoverlapping oligonucleotides prepared by standard methods and assembledinto a complete coding sequence. See, e.g., Edge, Nature, 292:756(1981); Nambair et al., Science, 223:1299 (1984); Jay et al., J. Biol.Chem., 259:6311 (1984).

Synthetic DNA sequences allow convenient construction of genes whichwill express CBP analogs or “muteins”. Alternatively, DNA encodingmuteins can be made by site-directed mutagenesis of native CBP genes orcDNAs, and muteins can be made directly using conventional polypeptidesynthesis.

A general method for site-specific incorporation of unnatural aminoacids into proteins is described in Christopher J. Noren, Spencer J.Anthony-Cahill, Michael C. Griffith, Peter G. Schultz, Science,244:182-188 (April 1989). This method may be used to create analogs withunnatural amino acids.

Recombinant Production of CBPs

Another feature of this invention is the expression of the DNA sequencesdisclosed herein. As is well known in the art, DNA sequences may beexpressed by operatively linking them to an expression control sequencein an appropriate expression vector and employing that expression vectorto transform an appropriate unicellular host.

Such operative linking of a DNA sequence of this invention to anexpression control sequence, of course, includes, if not already part ofthe DNA sequence, the provision of an initiation codon, ATG, in thecorrect reading frame upstream of the DNA sequence.

A wide variety of host/expression vector combinations may be employed inexpressing the DNA sequences of this invention. Useful expressionvectors, for example, may consist of segments of chromosomal,non-chromosomal and synthetic DNA sequences. Suitable vectors includederivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmidscol El, pCR1, pBR322, pMB9 and their derivatives, plasmids such as RP4;phage DNAS, e.g., the numerous derivatives of phage λ, e.g., NM989, andother phage DNA, e.g., M13 and filamentous single stranded phage DNA;yeast plasmids such as the 2μ plasmid or derivatives thereof; vectorsuseful in eukaryotic cells, such as vectors useful in insect ormammalian cells; vectors derived from combinations of plasmids and phageDNAs, such as plasmids that have been modified to employ phage DNA orother expression control sequences; and the like.

Any of a wide variety of expression control sequences—sequences thatcontrol the expression of a DNA sequence operatively linked to it—may beused in these vectors to express the DNA sequences of this invention.Such useful expression control sequences include, for example, the earlyor late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lacsystem, the trp system, the TAC system, the TRC system, the LTR system,the major operator and promoter regions of phage λ, the control regionsof fd coat protein, the promoter for 3-phosphoglycerate kinase or otherglycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), thepromoters of the yeast α-mating factors, and other sequences known tocontrol the expression of genes of prokaryotic or eukaryotic cells ortheir viruses, and various combinations thereof.

A wide variety of unicellular host cells are also useful in expressingthe DNA sequences of this invention. These hosts may include well knowneukaryotic and prokaryotic hosts, such as strains of E. coli,Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animalcells, such as CHO, R1.1, B-W and L-M cells, African Green Monkey kidneycells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells (e.g.,Sf9), and human cells and plant cells in tissue culture.

It will be understood that not all vectors, expression control sequencesand hosts will function equally well to express the DNA sequences ofthis invention. Neither will all hosts function equally well with thesame expression system. However, one skilled in the art will be able toselect the proper vectors, expression control sequences, and hostswithout undue experimentation to accomplish the desired expressionwithout departing from the scope of this invention. For example, inselecting a vector, the host must be considered because the vector mustfunction in it. The vector's copy number, the ability to control thatcopy number, and the expression of any other proteins encoded by thevector, such as antibiotic markers, will also be considered.

In selecting an expression control sequence, a variety of factors willnormally be considered. These include, for example, the relativestrength of the system, its controllability, and its compatibility withthe particular DNA sequence or gene to be expressed, particularly asregards potential secondary structures. Suitable unicellular hosts willbe selected by consideration of, e.g., their compatibility with thechosen vector, their secretion characteristics, their ability to foldproteins correctly, and their fermentation requirements, as well as thetoxicity to the host of the product encoded by the DNA sequences to beexpressed, and the ease of purification of the expression products.

Considering these and other factors a person skilled in the art will beable to construct a variety of vector/expression control sequence/hostcombinations that will express the DNA sequences of this invention onfermentation or in large scale animal culture.

It is further intended that CBP analogs may be prepared from nucleotidesequences of the protein complex/subunit derived within the scope of thepresent invention. Analogs, such as fragments, may be produced, forexample, by pepsin digestion of CBP or bacterial material. Otheranalogs, such as muteins, can be produced by standard site-directedmutagenesis of CBP coding sequences. Analogs exhibiting “choline-bindingactivity” such as small molecules, whether functioning as promoters orinhibitors, may be identified by known in vivo and/or in vitro assays.

Antibodies to CBPs

As noted above, the CBPs of the invention, whether obtained bypurification from bacterial sources or recombinantly, can be used togenerate antibodies for diagnosis and therapy, as set forth in detailbelow. Thus, preferred CBPs of the invention are antigenic, and morepreferably immunogenic.

A molecule is “antigenic” when it is capable of specifically interactingwith an antigen recognition molecule of the immune system, such as animmunoglobulin (antibody) or T cell antigen receptor. An antigenicpolypeptide contains at least about 5, and preferably at least about 10,amino acids. An antigenic portion of a molecule can be that portion thatis immunodominant for antibody or T cell receptor recognition, or it canbe a portion used to generate an antibody to the molecule by conjugatingthe antigenic portion to a carrier molecule for immunization. A moleculethat is antigenic need not be itself immunogenic, i.e., capable ofeliciting a humoral immune response without a carrier.

An “antibody” for purposes of this invention is any immunoglobulin,including antibodies and fragments thereof, that binds a specificepitope on a CBP. The term encompasses polyclonal, monoclonal, andchimeric antibodies, the last mentioned described in further detail inU.S. Pat. Nos. 4,816,397 and 4,816,567.

An “antibody combining site” is that structural portion of an antibodymolecule comprised of heavy and light chain variable and hypervariableregions that specifically binds antigen.

The phrase “antibody molecule” in its various grammatical forms as usedherein contemplates both an intact immunoglobulin molecule and animmunologically active portion of an immunoglobulin molecule.

According to the invention, CBP(s) produced recombinantly, by chemicalsynthesis, or purified from the natural bacterial source, and fragmentsor other derivatives or analogs thereof, including fusion proteins, maybe used as an immunogen to generate anti-CBP antibodies. Such antibodiesinclude but are not limited to polyclonal, monoclonal, chimeric, singlechain, Fab fragments, and an Fab expression library.

Various procedures known in the art may be used for the production ofpolyclonal antibodies to CBPs or derivatives or analogs thereof (see,e.g., Antibodies—A Laboratory Manual, Harlow and Lane, eds., Cold SpringHarbor Laboratory Press: Cold Spring Harbor, N.Y., 1988). For theproduction of antibody, various host animals can be immunized byinjection with CBP(s), or a derivative (e.g., fragment or fusionprotein) thereof, including but not limited to rabbits, mice, rats,sheep, goats, etc. In one embodiment, the CBP or fragment thereof can beconjugated to an immunogenic carrier, e.g., bovine serum albumin (BSA)or keyhole limpet hemocyanin (KLH). Various adjuvants may be used toincrease the immunological response, depending on the host species.

For preparation of monoclonal antibodies directed toward the CBP, orfragment, analog, or derivative thereof, any technique that provides forthe production of antibody molecules by continuous cell lines in culturemay be used (see, e.g., Antibodies—A Laboratory Manual, Harlow and Lane,eds., Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y.,1988). These include but are not limited to the hybridoma techniqueoriginally developed by Kohler and Milstein (1975, Nature 256:495-497),as well as the trioma technique, the human B-cell hybridoma technique(Kozbbr et al., 1983, Immunology Today 4:72), and the EBV-hybridomatechnique to produce human monoclonal antibodies (Cole et al., 1985, inMonoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96).In an additional embodiment of the invention, monoclonal antibodies canbe produced in germ-free animals utilizing recent technology(PCT/US90/02545). According to the invention, human antibodies may beused and can be obtained by using human hybridomas (Cote et al., 1983,Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030) or by transforming human Bcells with EBV virus in vitro (Cole et al., 1985, in MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, pp. 77-96). In fact,according to the invention, techniques developed for the production of“chimeric antibodies” (Morrison et al., 1984, J. Bacteriol. 159-870;Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature314:452-454) by splicing the genes from a mouse antibody moleculespecific for a CBP together with genes from a human antibody molecule ofappropriate biological activity can be used; such antibodies are withinthe scope of this invention. Such human or humanized chimeric antibodiesare preferred for use in therapy of human diseases or disorders(described infra), since the human or humanized antibodies are much lesslikely than xenogenic antibodies to induce an immune response, inparticular an allergic response, themselves.

According to the invention, techniques described for the production ofsingle chain antibodies (U.S. Pat. No. 4,946,778) can be adapted toproduce CBP-specific single chain antibodies. An additional embodimentof the invention utilizes the techniques described for the constructionof Fab expression libraries (Huse et al., 1989, Science 246:1275-1281)to allow rapid and easy identification of monoclonal Fab fragments withthe desired specificity for a CBP, or its derivatives, or analogs.

Antibody fragments which contain the idiotype of the antibody moleculecan be generated by known techniques. For example, such fragmentsinclude but are not limited to: the F(ab′)₂ fragment which can beproduced by pepsin digestion of the antibody molecule; the Fab′fragments which can be generated by reducing the disulfide bridges ofthe F(ab′)₂ fragment, and the Fab fragments which can be generated bytreating the antibody molecule with papain and a reducing agent.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g., radioimmunoassay,ELISA (enzyme-linked immunosorbant assay), “sandwich” immunoassays,immunoradiometric assays, gel diffusion precipitin reactions,immunodiffusion assays, in situ immunoassays (using colloidal gold,enzyme or radioisotope labels, for example), western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, theprimary antibody is detected by detecting binding of a secondaryantibody or reagent to the primary antibody. In a further embodiment,the secondary antibody is labeled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention. For example, to select antibodies which recognize aspecific epitope of a CBP, one may assay generated hybridomas for aproduct which binds to a CBP fragment containing such epitope. Forselection of an antibody specific to a CBP from a particular strain ofGram positive bacteria, particularly pneumococcus, one can select on thebasis of positive binding with CBP expressed by or isolated from cellsof that strain of bacteria.

The foregoing antibodies can be used in methods known in the artrelating to the localization and activity of the CBP, e.g., for Westernblotting, imaging CBP polypeptide in situ, measuring levels thereof inappropriate physiological samples, etc.

In a specific embodiment, antibodies that agonize or antagonize theactivity of CBP can be generated. Such antibodies can be tested usingthe assays described supra for characterizing CBPs.

The term “adjuvant” refers to a compound or mixture that enhances theimmune response to an antigen. An adjuvant can serve as a tissue depotthat slowly releases the antigen and also as a lymphoid system activatorthat non-specifically enhances the immune response (Hood et al.,Immunology, Second Ed., 1984, Benjamin/Cummings: Menlo Park, Calif., p.384). Often, a primary challenge with an antigen alone, in the absenceof an adjuvant, will fail to elicit a humoral or cellular immuneresponse. Adjuvants include, but are not limited to, complete Freund'sadjuvant, incomplete Freund's adjuvant, saponin, mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum. Preferably, the adjuvant is pharmaceutically acceptable.

Vaccination and Passive Immune Therapy

Active immunity against Gram positive bacteria, particularlypneumococcus, can be induced by immunization (vaccination) with animmunogenic amount of an CBP, or an antigenic derivative or fragmentthereof, and an adjuvant, wherein the CBP, or antigenic derivative orfragment thereof, is the antigenic component of the vaccine.

The CBP alone or conjugated to a capsule or capsules cannot causebacterial infection, and the active immunity elicited by vaccinationwith the protein according to the present invention can result in bothan immediate immune response and in immunological memory, and thusprovide long-term protection against infection by the bacterium. TheCBPs of the present invention, or antigenic fragments thereof, can beprepared in an admixture with an adjuvant to prepare a vaccine.Preferably, the CBP, or derivative or fragment thereof, used as theantigenic component of the vaccine is an adhesin. More preferably, theCBP, or derivative or fragment thereof, used as the antigenic of thevaccine is an antigen common to all or many strains of a species of Grampositive bacteria, or common to closely related species of bacteria.Most preferably, the antigenic component of the vaccine is an adhesinthat is a common antigen.

Selection of an adjuvant depends on the subject to be vaccinated.Preferably, a pharmaceutically acceptable adjuvant is used. For example,a vaccine for a human should avoid oil or hydrocarbon emulsionadjuvants, including complete and incomplete Freund's adjuvant. Oneexample of an adjuvant suitable for use with humans is alum (aluminagel). A vaccine for an animal, however, may contain adjuvants notappropriate for use with humans.

An alternative to a traditional vaccine comprising an antigen and anadjuvant involves the direct in vivo introduction of DNA encoding theantigen into tissues of a subject for expression of the antigen by thecells of the subject's tissue. Such vaccines are termed herein “nucleicacid-based vaccines.” Since the CBP gene by definition contains a signalsequence, expression of the gene in cells of the tissue results insecretion of membrane association of the expressed protein.Alternatively, the expression vector can be engineered to contain anautologous signal sequence instead of the CBP signal sequence. Forexample, a naked DNA vector (see, e.g., Ulmer et al., 1993, Science259:1745-1749), a DNA vector transporter (e.g., Wu et al., 1992, J.Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem.263:14621-14624; Hartmut et al., Canadian Patent Application No.2,012,311, filed Mar. 15, 1990), or a viral vector containing thedesired CBP gene can be injected into tissue. Suitable viral vectorsinclude tetroviruses that are packaged in cells with amphotropic hostrange (see Miller, 1990, Human Gene Ther. 1:5-14; Ausubel et al.,Current Protocols in Molecular Biology, § 9), and attenuated ordefective DNA virus, such as but not limited to herpes simplex virus(HSV) (see, e.g., Kaplitt et al., 1991, Molec. Cell. Neurosci.2:320-330), papillomavirus, Epstein Barr virus (EBV), adenovirus (see,e.g., Stratford-Perricaudet et al., 1992, J. Clin. Invest. 90:626-630),adeno-associated virus (AAV) (see, e.g., Samulski et al., 1987, J.Virol. 61:3096-3101; Samulski et al., 1989, J. Virol. 63:3822-3828), andthe like. Defective viruses, which entirely or almost entirely lackviral genes, are preferred. Defective virus is not infective afterintroduction into a cell.

Vectors containing the nucleic acid-based vaccine of the invention canbe introduced into the desired host by methods known in the art, e.g.,transfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, lipofection(lysosome fusion), use of a gene gun, or a DNA vector transporter (see,e.g., Wu et al., 1992, J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J.Biol. Chem. 263:14621-14624; Hartmut et al., Canadian Patent ApplicationNo. 2,012,311, filed Mar. 15, 1990).

Either vaccine of the invention, i.e., a vaccines comprising an CBPantigen or antigenic derivative or fragment thereof, or an CBP nucleicacid vaccine, can be administered via any parenteral route, includingbut not limited to intramuscular, intraperitoneal, intravenous, and thelike. Preferably, since the desired result of vaccination is toelucidate an immune response to the antigen, and thereby to thepathogenic organism, administration directly, or by targeting or choiceof a viral vector, indirectly, to lymphoid tissues, e.g., lymph nodes orspleen. Since immune cells are continually replicating, they are idealtarget for retroviral vector-based nucleic acid vaccines, sinceretroviruses require replicating cells.

Passive immunity can be conferred to an animal subject suspected ofsuffering an infection with a Gram positive bacterium, preferablystreptococcal, and more preferably pneumoccal, by administeringantiserum, polyclonal antibodies, or a neutralizing monoclonal antibodyagainst a choline binding protein of the invention to the patient.Although passive immunity does not confer long term protection, it canbe a valuable tool for the treatment of a bacterial infection of asubject who has not been vaccinated. Passive immunity is particularlyimportant for the treatment of antibiotic resistant strains of Grampositive bacteria, since no other therapy may be available. Preferably,the antibodies administered for passive immune therapy are autologousantibodies. For example, if the subject is a human, preferably theantibodies are of human origin or have been “humanized,” in order tominimize the possibility of an immune response against the antibodies.In a specific example, infra, passive immunity completely protectsagainst lethal bacterial infection.

An analogous therapy to passive immunization is administration of anamount of an CBP protein adhesin sufficient to inhibit adhesion of thebacterium to its target cell. The required amount can be determined byone of ordinary skill using standard techniques.

The active or passive vaccines of the invention, or the administrationof an adhesin, can be used to protect an animal subject from infectionof a Gram positive bacteria, preferably streptococcus, and morepreferably, pneumococcus. Thus, a vaccine of the invention can be usedin birds, such as chickens, turkeys, and pets; in mammals, preferably ahuman, although the vaccines of the invention are contemplated for usein other mammalian species, including but not limited to domesticatedanimals (canine and feline); farm animals (bovine, ovine, equine,caprine, porcine, and the like); rodents; and undomesticated animals.

Diagnosis of a Gram Positive Bacterial Infection

The antibodies of the present invention that can be generated againstthe CBPs from Gram positive bacteria are valuable reagents for thediagnosis of an infection with a Gram positive microorganism,particularly a pneumococcus. Presently, diagnosis of infection with aGram positive bacterium is difficult. According to the invention, thepresence of Gram positive bacteria in a sample from a subject suspectedof having an infection with a Gram positive bacterium can be detected bydetecting binding of an antibody to an CBP to bacteria in or from thesample. In one aspect of the invention, the antibody can be specific fora unique strain or a limited number of strains of the bacterium, thusallowing for diagnosis of infection with that particular strain (orstrains). Alternatively, the antibody can be specific for many or allstrains of a bacterium, thus allowing for diagnosis of infection withthat species.

Diagnosis of infection with a Gram positive bacterium can use anyimmunoassay format known in the art, as desired. Many possibleimmunoassay formats are described in the section entitled “Antibodies toCBPs.” The antibodies can be labeled for detection in vitro, e.g., withlabels such as enzymes, fluorophores, chromophores, radioisotopes, dyes,colloidal gold, latex particles, and chemiluminescent agents.Alternatively, the antibodies can be labeled for detection in vivo,e.g., with radioisotopes (preferably technetium or iodine); magneticresonance shift reagents (such as gadolinium and manganese); orradio-opaque reagents.

In specific embodiments, the presence of CBP in or on cells can beascertained by the usual immunological procedures applicable to suchdeterminations. A number of useful procedures are known. The proceduresand their application are all familiar to those skilled in the art andaccordingly may be utilized within the scope of the present invention.For example, a “competitive” procedure is described in U.S. Pat. Nos.3,654,090 and 3,850,752. A “sandwich” procedure, is described in U.S.Pat. Nos. RE 31,006 and 4,016,043. Still other procedures are known suchas the “double antibody,” or “DASP” procedure.

In each instance, the CBP forms complexes with one or more antibody(ies)or binding partners and one member of the complex is labeled with adetectable label. The fact that a complex has formed and, if desired,the amount thereof, can be determined by known methods applicable to thedetection of labels.

The labels most commonly employed for these studies are radioactiveelements, enzymes, chemicals which fluoresce when exposed to ultravioletlight, and others.

A number of fluorescent materials are known and can be utilized aslabels. These include, for example, fluorescein, rhodamine, auramine,Texas Red, AMCA blue and Lucifer Yellow. A particular detecting materialis anti-rabbit antibody prepared in goats and conjugated withfluorescein through an isothiocyanate.

The CBP or its binding partner(s) can also be labeled with a radioactiveelement or with an enzyme. The radioactive label can be detected by anyof the currently available counting procedures. The preferred isotopemay be selected from ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe,⁹⁰Y, ¹²⁵I, ¹³¹I, and ¹⁸⁶Re.

Enzyme labels are likewise useful, and can be detected by any of thepresently utilized colorimetric, spectrophotometricfluorospectrophotometric, amperometric or gasometric techniques. Theenzyme is conjugated to the selected particle by reaction with bridgingmolecules such as carbodiimides, diisocyanates, glutaraldehyde and thelike. Many enzymes which can be used in these procedures are known andcan be utilized. The preferred are peroxidase, β-glucuronidase,β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plusperoxidase and alkaline phosphatase. U.S. Pat. Nos. 3,654,090;3,850,752; and 4,016,043 are referred to by way of example for theirdisclosure of alternate labeling material and methods.

In a further embodiment of this invention, commercial test kits suitablefor use by a medical specialist may be prepared to determine thepresence or absence of predetermined binding activity or predeterminedbinding activity capability to suspected target cells. In accordancewith the testing techniques discussed above, one class of such kits willcontain at least the labeled CBPor its binding partner, for instance anantibody specific thereto, and directions, of course, depending upon themethod selected, e.g., “competitive,” “sandwich,” “DASP” and the like.The kits may also contain peripheral reagents such as buffers,stabilizers, etc.

Accordingly, a test kit may be prepared for the demonstration of thepresence or capability of cells for predetermined bacterial bindingactivity, comprising:

-   -   (a) a predetermined amount of at least one labeled        immunochemically reactive component obtained by the direct or        indirect attachment of the present CBP factor or a specific        binding partner thereto, to a detectable label;    -   (b) other reagents; and    -   (c) directions for use of said kit.

More specifically, the diagnostic test kit may comprise:

-   -   (a) a known amount of the CBP as described above (or a binding        partner) generally bound to a solid phase to form an        immunosorbent, or in the alternative, bound to a suitable tag,        or plural such end products, etc. (or their binding partners)        one of each;    -   (b) if necessary, other reagents; and    -   (c) directions for use of said test kit.

In a further variation, the test kit may be prepared and used for thepurposes stated above, which operates according to a predeterminedprotocol (e.g. “competitive,” “sandwich,” “double antibody,” etc.), andcomprises:

-   -   (a) a labeled component which has been obtained by coupling the        CBP to a detectable label;    -   (b) one or more additional immunochemical reagents of which at        least one reagent is a ligand or an immobilized ligand, which        ligand is selected from the group consisting of:        -   (i) a ligand capable of binding with the labeled component            (a);        -   (ii) a ligand capable of binding with a binding partner of            the labeled component (a);        -   (iii) a ligand capable of binding with at least one of the            component(s) to be determined; and        -   (iv) a ligand capable of binding with at least one of the            binding partners of at least one of the component(s) to be            determined; and    -   (c) directions for the performance of a protocol for the        detection and/or determination of one or more components of an        immunochemical reaction between the CBP and a specific binding        partner thereto.

Alternatively, the nucleic acids and sequences thereof of the inventioncan be used in the diagnosis of infection with a Gram positivebacterium. For example, the CBP genes or hybridizable fragments thereofcan be used for in situ hybridization with a sample from a subjectsuspected of harboring an infection of Gram positive bacteria. Inanother embodiment, specific gene segments of a Gram positive bacteriumcan be identified using PCR amplification with probes based on the CBPgenes of the invention. In one aspect of the invention, thehybridization with a probe or with the PCR primers can be performedunder stringent conditions, or with a sequence specific for a uniquestrain or a limited number of strains of the bacterium, or both, thusallowing for diagnosis of infection with that particular strain (orstrains). Alternatively, the hybridization can be under less stringentconditions, or the sequence may be homologous in any or all strains of abacterium, thus allowing for diagnosis of infection with that species.

Therapeutic Compositions and Vaccines Comprising CBPs

As noted above, the present invention provides therapeutic compositionscomprising antibodies to CBPs (i.e., passive immune therapy), anti-CBPvaccines (whether comprised of CBPs in an adjuvant, or a nucleic acidvaccine), CBPs to compete with bacterial CBPs for pathogenic activities,such as adherence to host cells, peptides, such as Fn5, or antibodies toFn5. Preferably, any such composition is a pharmaceutical compositioncomprising the active component (antibody, vaccine, or CBP) in apharmaceutically acceptable carrier.

The preparation of therapeutic compositions which contain an activecomponent is well understood in the art. Typically, such compositionsare prepared as injectables, either as liquid solutions or suspensions,however, solid forms suitable for solution in, or suspension in, liquidprior to injection can also be prepared. The preparation can also beemulsified. The active therapeutic ingredient is often mixed withexcipients which are pharmaceutically acceptable and compatible with theactive ingredient. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol, or the like and combinations thereof. Inaddition, if desired, the composition can contain minor amounts ofauxiliary substances such as wetting or emulsifying agents, pH bufferingagents which enhance the effectiveness of the active ingredient.

A active component can be formulated into the therapeutic composition asneutralized pharmaceutically acceptable salt forms. Pharmaceuticallyacceptable salts include the acid addition salts (formed with the freeamino groups of the polypeptide or antibody molecule) and which areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed from the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, 2-ethylamino ethanol,histidine, procaine, and the like.

A composition comprising “A” (where “A” is a single protein, DNAmolecule, vector, etc.) is substantially free of “B” (where “B”comprises one or more contaminating proteins, DNA molecules, vectors,etc.) when at least about 75% by weight of the proteins, DNA, vectors(depending on the category of species to which A and B belong) in thecomposition is “A”. Preferably, “A” comprises at least about 90% byweight of the A+B species in the composition, most preferably at leastabout 99% by weight.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human. Preferably, asused herein, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the compound isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water or aqueous solution saline solutions and aqueousdextrose and glycerol solutions are preferably employed as carriers,particularly for injectable solutions. Suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E. W. Martin.

The phrase “therapeutically effective amount” is used herein to mean anamount sufficient to reduce by at least about 15 percent, preferably byat least 50 percent, more preferably by at least 90 percent, and mostpreferably prevent, a clinically significant deficit in the activity,function and response of the host. Alternatively, a therapeuticallyeffective amount is sufficient to cause an improvement in a clinicallysignificant condition in the host. In the context of the presentinvention, a deficit in the response of the host is evidenced bycontinuing or spreading bacterial infection. An improvement in aclinically significant condition in the host includes a decrease inbacterial load, clearance of bacteria from colonized host cells,reduction in fever or inflammation associated with infection, or areduction in any symptom associated with the bacterial infection.

According to the invention, the component or components of a therapeuticcomposition of the invention may be introduced parenterally,transmucosally, e.g., orally, nasally, pulmonarilly, or rectally, ortransdermally. Preferably, administration is parenteral, e.g., viaintravenous injection, and also including, but is not limited to,intra-arteriole, intramuscular, intradermal, subcutaneous,intraperitoneal, intraventricular, and intracranial administration. Oralor pulmonary delivery may be preferred to activate mucosal immunity;since pneumococci generally colonize the nasopharyngeal and pulmonarymucosa, mucosal immunity may be a particularly effective preventivetreatment. The term “unit dose” when used in reference to a therapeuticcomposition of the present invention refers to physically discrete unitssuitable as unitary dosage for humans, each unit containing apredetermined quantity of active material calculated to produce thedesired therapeutic effect in association with the required diluent;i.e., carrier, or vehicle.

In another embodiment, the active compound can be delivered in avesicle, in particular a liposome (see Langer, Science 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss, N.Y., pp. 353-365(1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid).

In yet another embodiment, the therapeutic compound can be delivered ina controlled release system. For example, the polypeptide may beadministered using intravenous infusion, an implantable osmotic pump, atransdermal patch, liposomes, or other modes of administration. In oneembodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit.Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980);Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment,polymeric materials can be used (see Medical Applications of ControlledRelease, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974);Controlled Drug Bioavailability, Drug Product Design and Performance,Smolen and Ball (eds.), Wiley, N.Y. (1984); Ranger and Peppas, J.Macrbmol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al.,Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989);Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment,a controlled release system can be placed in proximity of thetherapeutic target, i.e., the brain, thus requiring only a fraction ofthe systemic dose (see, e.g., Goodson, in Medical Applications ofControlled Release, supra, vol. 2, pp. 115-138 (1984)). Preferably, acontrolled release device is introduced into a subject in proximity ofthe site of inappropriate immune activation or a tumor.

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

A subject in whom administration of an active component as set forthabove is an effective therapeutic regiment for a bacterial infection ispreferably a human, but can be any animal. Thus, as can be readilyappreciated by one of ordinary skill in the art, the methods andpharmaceutical compositions of the present invention are particularlysuited to administration to any animal, particularly a mammal andincluding, but by no means limited to, domestic animals, such as felineor canine subjects, farm animals, such as but not limited to bovine,equine, caprine, ovine, and porcine subjects, wild animals (whether inthe wild or in a zoological garden), research animals, such as mice,rats, rabbits, goats, sheep, pigs, dogs, cats, etc., i.e., forveterinary medical use.

In the therapeutic methods and compositions of the invention, atherapeutically effective dosage of the active component is provided. Atherapeutically effective dosage can be determined by the ordinaryskilled medical worker based on patient characteristics (age, weight,sex, condition, complications, other diseases, etc.), as is well knownin the art. Furthermore, as further routine studies are conducted, morespecific information will emerge regarding appropriate dosage levels fortreatment of various conditions in various patients, and the ordinaryskilled worker, considering the therapeutic context, age and generalhealth of the recipient, is able to ascertain proper dosing. Generally,for intravenous injection or infusion, dosage may be lower than forintraperitoneal, intramuscular, or other route of administration. Thedosing schedule may vary, depending on the circulation half-life, andthe formulation used. The compositions are administered in a mannercompatible with the dosage formulation in the therapeutically effectiveamount. Precise amounts of active ingredient required to be administereddepend on the judgment of the practitioner and are peculiar to eachindividual. However, suitable dosages may range from about 0.1 to 20,preferably about 0.5 to about 10, and more preferably one to several,milligrams of active ingredient per kilogram body weight of individualper day and depend on the route of administration. Suitable regimes forinitial administration and booster shots are also variable, but aretypified by an initial administration followed by repeated doses at oneor more hour intervals by a subsequent injection or otheradministration. Alternatively, continuous intravenous infusionsufficient to maintain concentrations of ten nanomolar to ten micromolarin the blood are contemplated.

Administration with other compounds. For treatment of a bacterialinfection, one may administer the present active component inconjunction with one or more pharmaceutical compositions used fortreating bacterial infection, including but not limited to (1)antibiotics; (2) soluble carbohydrate inhibitors of bacterial adhesion;(3) other small molecule inhibitors of bacterial adhesion; (4)inhibitors of bacterial metabolism, transport, or transformation; (5)stimulators of bacterial lysis, or (6) anti-bacterial antibodies orvaccines directed at other bacterial antigens. Other potential activecomponents include anti-inflammatory agents, such as steroids andnon-steroidal anti-inflammatory drugs. Administration may besimultaneous (for example, administration of a mixture of the presentactive component and an antibiotic), or may be in serriatim.

Accordingly, in specific embodiment, the therapeutic compositions mayfurther include an effective amount of the active component, and one ormore of the following active ingredients: an antibiotic, a steroid, etc.Exemplary formulations are given below:

Formulations

Ingredient mg/ml Intravenous Formulation I cefotaxime 250.0 CBP 10.0dextrose USP 45.0 sodium bisulfite USP 3.2 edetate disodium USP 0.1water for injection q.s.a.d. 1.0 ml Intravenous Formulation IIampicillin 250.0 CBP 10.0 sodium bisulfite USP 3.2 disodium edetate USP0.1 water for injection q.s.a.d. 1.0 ml Intravenous Formulation IIIgentamicin (charged as sulfate) 40.0 CBP 10.0 sodium bisulfite USP 3.2disodium edetate USP 0.1 water for injection q.s.a.d. 1.0 ml IntravenousFormulation IV CBP 10.0 dextrose USP 45.0 sodium bisulfite USP 3.2edetate disodium USP 0.1 water for injection q.s.a.d. 1.0 ml IntravenousFormulation V CBP antagonist 5.0 sodium bisulfite USP 3.2 disodiumedetate USP 0.1 water for injection q.s.a.d. 1.0 ml

As used herein, “pg” means picogram, “ng” means nanogram, “ug” or “μg”mean microgram, “mg” means milligram, “ul” or “μl” mean microliter, “ml”means milliliter, “l” means liter.

Thus, in a specific instance where it is desired to reduce or inhibitthe infection resulting from CBP-mediated binding of bacteria to a hostcell, CBP or an antibody thereto, or a ligand thereof or an antibody tothat ligand, such as Fn5, could be introduced to block the interactionof CBP present on bacteria with the host cell.

As discussed earlier, the CBPs or antibodies thereto may be prepared inpharmaceutical compositions, with a suitable carrier and at a strengtheffective for administration by various means to a patient experiencingan adverse medical condition associated with specific bacterialinfection for the treatment thereof. A variety of administrativetechniques may be utilized, among them parenteral techniques such assubcutaneous, intravenous and intraperitoneal injections,catheterizations and the like. Average quantities of the CBPs or theirsubunits may vary and in particular should be based upon therecommendations and prescription of a qualified physician orveterinarian.

Pulmonary Delivery

Also contemplated herein is pulmonary delivery of the present adhesioninhibitory agent (or derivatives thereof), of the invention, selectedfrom the group consisting of a choline binding protein, an antibody to acholine binding protein, an enolase, hindered cationic small molecules(such as lysine, choline, arginine, etc.), the peptide WQPPRARI (SEQ IDNO:11), and antibody specific for an epitope having the amino acidsequence WQPPRARI (SEQ ID NO:11). The adhesion inhibitory agent (orderivative) is delivered to the lungs of a mammal, where it caninterfere with bacterial, i.e., streptococcal, and preferablypneumococcal binding to host cells. In a specific embodiment, such anadhesion inhibitory agent inhibits binding of the streptococcal enolaseto fibronectin. Other reports of preparation of proteins for pulmonarydelivery are found in the art [Adjei et al. Pharmaceutical Research,7:565-569 (1990); Adjei et al., International Journal of Pharmaceutics,63:135-144 (1990) (leuprolide acetate); Braquet et al., Journal ofCardiovascular Pharmacology, 13(suppl. 5):143-146 (1989) (endothelin-1);Hubbard et al., Annals of Internal Medicine, Vol. III, pp. 206-212(1989) (α1-antitrypsin); Smith et al., J. Clin. Invest. 84:1145-1146(1989) (α-1-proteinase); Oswein et al., “Aerosolization of Proteins”,Proceedings of Symposium on Respiratory Drug Delivery II, Keystone,Colo., March, (1990) (recombinant human growth hormone); Debs et al., J.Immunol. 140:3482-3488 (1988) (interferon-γ and tumor necrosis factoralpha); Platz et al., U.S. Pat. No. 5,284,656 (granulocyte colonystimulating factor)]. A method and composition for pulmonary delivery ofdrugs is described in U.S. Pat. No. 5,451,569, issued Sep. 19, 1995 toWong et al.

Contemplated for use in the practice of this invention are a wide rangeof mechanical devices designed for pulmonary delivery of therapeuticproducts, including but not limited to nebulizers, metered doseinhalers, and powder inhalers, all of which are familiar to thoseskilled in the art. With regard to construction of the delivery device,any form of aerosolization known in the art, including but not limitedto spray bottles, nebulization, atomization or pump aerosolization of aliquid formulation, and aerosolization of a dry powder formulation, canbe used in the practice of the invention.

Some specific examples of commercially available devices suitable forthe practice of this invention are the Ultravent nebulizer, manufacturedby Mallinckrodt, Inc., St. Louis, Mo.; the Acorn II nebulizer,manufactured by Marquest Medical Products, Englewood, Colo.; theVentolin metered dose inhaler, manufactured by Glaxo Inc., ResearchTriangle Park, N.C.; and the Spinhaler powder inhaler, manufactured byFisons Corp., Bedford, Mass.

All such devices require the use of formulations suitable for thedispensing of adhesion inhibitory agent (or derivative). Typically, eachformulation is specific to the type of device employed and may involvethe use of an appropriate propellant material, in addition to the usualdiluents, adjuvants and/or carriers useful in therapy. Also, the use ofliposomes, microcapsules or microspheres, inclusion complexes, or othertypes of carriers is contemplated. Chemically modified adhesioninhibitory agent may also be prepared in different formulationsdepending on the type of chemical modification or the type of deviceemployed.

Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise adhesion inhibitory agent (orderivative) dissolved in water at a concentration of about 0.1 to 25 mgof biologically active adhesion inhibitory agent per ml of solution. Theformulation may also include a buffer and a simple sugar (e.g., foradhesion inhibitory agent stabilization and regulation of osmoticpressure). The nebulizer formulation may also contain a surfactant, toreduce or prevent surface induced aggregation of the adhesion inhibitoryagent caused by atomization of the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device will generallycomprise a finely divided powder containing the adhesion inhibitoryagent (or derivative) suspended in a propellant with the aid of asurfactant. The propellant may be any conventional material employed forthis purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, ahydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane,dichlorodifluoromethane, dichlorotetrafluoroethanol, and1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactantsinclude sorbitan trioleate and soya lecithin. Oleic acid may also beuseful as a surfactant.

The liquid aerosol formulations contain adhesion inhibitory agent and adispersing agent in a physiologically acceptable diluent. The dry powderaerosol formulations of the present invention consist of a finelydivided solid form of adhesion inhibitory agent and a dispersing agent.With either the liquid or dry powder aerosol formulation, theformulation must be aerosolized. That is, it must be broken down intoliquid or solid particles in order to ensure that the aerosolized doseactually reaches the mucous membranes of the nasal passages or the lung.The term “aerosol particle” is used herein to describe the liquid orsolid particle suitable for nasal or pulmonary administration, i.e.,that will reach the mucous membranes. Other considerations, such asconstruction of the delivery device, additional components in theformulation, and particle characteristics are important. These aspectsof pulmonary administration of a drug are well known in the art, andmanipulation of formulations, aerosolization means and construction of adelivery device require at most routine experimentation by one ofordinary skill in the art.

In a particular embodiment, the mass median dynamic diameter will be 5micrometers or less in order to ensure that the drug particles reach thelung alveoli [Wearley, L. L., Crit. Rev. in Ther. Drug Carrier Systems8:333 (1991)].

Systems of aerosol delivery, such as the pressurized metered doseinhaler and the dry powder inhaler are disclosed in Newman, S. P.,Aerosols and the Lung, Clarke, S. W. and Davia, D. editors, pp. 197-22and can be used in connection with the present invention.

In a further embodiment, as discussed in detail infra, an aerosolformulation of the present invention can include other therapeuticallyor pharmacologically active ingredients in addition to adhesioninhibitory agent, such as but not limited to an antibiotic, a steroid, anon-steroidal anti-inflammatory drug, etc.

Liquid Aerosol Formulations. The present invention provides aerosolformulations and dosage forms for use in treating subjects sufferingfrom bacterial, e.g., streptococcal, in particularly pneumococcal,infection. In general such dosage forms contain adhesion inhibitoryagent in a pharmaceutically acceptable diluent. Pharmaceuticallyacceptable diluents include but are not limited to sterile water,saline, buffered saline, dextrose solution, and the like. In a specificembodiment, a diluent that may be used in the present invention or thepharmaceutical formulation of the present invention is phosphatebuffered saline, or a buffered saline solution generally between the pH7.0-8.0 range, or water.

The liquid aerosol formulation of the present invention may include, asoptional ingredients, pharmaceutically acceptable carriers, diluents,solubilizing or emulsifying agents, surfactants and excipients.

The formulation may include a carrier. The carrier is a macromoleculewhich is soluble in the circulatory system and which is physiologicallyacceptable where physiological acceptance means that those of skill inthe art would accept injection of said carrier into a patient as part ofa therapeutic regime. The carrier preferably is relatively stable in thecirculatory system with an acceptable plasma half life for clearance.Such macromolecules include but are not limited to Soya lecithin, oleicacid and sorbitan trioleate, with sorbitan trioleate preferred.

The formulations of the present embodiment may also include other agentsuseful for pH maintenance, solution stabilization, or for the regulationof osmotic pressure. Examples of the agents include but are not limitedto salts, such as sodium chloride, or potassium chloride, andcarbohydrates, such as glucose, galactose or mannose, and the like.

The present invention further contemplates liquid aerosol formulationscomprising adhesion inhibitory agent and another therapeuticallyeffective drug, such as an antibiotic, a steroid, a non-steroidalanti-inflammatory drug, etc.

Aerosol Dry Powder Formulations. It is also contemplated that thepresent aerosol formulation can be prepared as a dry powder formulationcomprising a finely divided powder form of adhesion inhibitory agent anda dispersant.

Formulations for dispensing from a powder inhaler device will comprise afinely divided dry powder containing adhesion inhibitory agent (orderivative) and may also include a bulking agent, such as lactose,sorbitol, sucrose, or mannitol in amounts which facilitate dispersal ofthe powder from the device, e.g., 50 to 90% by weight of theformulation. The adhesion inhibitory agent (or derivative) should mostadvantageously be prepared in particulate form with an average particlesize of less than 10 mm (or microns), most preferably 0.5 to 5 mm, formost effective delivery to the distal lung.

In another embodiment, the dry powder formulation can comprise a finelydivided dry powder containing adhesion inhibitory agent, a dispersingagent and also a bulking agent. Bulking agents useful in conjunctionwith the present formulation include such agents as lactose, sorbitol,sucrose, or mannitol, in amounts that facilitate the dispersal of thepowder from the device.

The present invention further contemplates dry powder formulationscomprising adhesion inhibitory agent and another therapeuticallyeffective drug, such as an antibiotic, a steroid, a non-steroidalanti-inflammatory drug, etc.

Identification of Small Molecule Antagonists of CBPs

Identification and isolation of a gene encoding a choline bindingprotein of the invention provides for expression of the protein inquantities greater than can be isolated from natural sources, or inindicator cells that are specially engineered to indicate the activityof a CBP expressed after transfection or transformation of the cells.According, in addition to rational design of agonists and antagonistsbased on the structure of a choline binding protein, the presentinvention contemplates an alternative method for identifying specificligands of a choline binding-protein using various screening assaysknown in the art. Examples of small molecule antagonists of CBPs includelysine, choline, the pentapeptide having SEQ ID NO:11; it is likely thatanalogs of these types of molecules, such as arginine, will also inhibitbacterial adhesion to host tissue, particularly fibronectin. Thus, thepresent invention generally provides a small molecule CBP bindingantagonist, which is a hindered cationic molecule, preferably a hinderedamine.

Any screening technique known in the art can be used to screen forcholine binding protein agonists or antagonists. The present inventioncontemplates screens for small molecule ligands or ligand analogs andmimics, as well as screens for natural ligands that bind to and agonizeor antagonize activities of choline binding protein in vivo,particularly adhesion of bacteria to host cells or tissues mediated bythe choline binding protein.

Knowledge of the primary sequence of the CBP, and the similarity of thatsequence with proteins of known function, can provide an initial clue asthe inhibitors or antagonists of the protein. Identification andscreening of antagonists is further facilitated by determiningstructural features of the protein, e.g., using X-ray crystallography,neutron diffraction, nuclear magnetic resonance spectrometry, and othertechniques for structure determination. These techniques provide for therational design or identification of agonists and antagonists.

Another approach uses recombinant bacteriophage to produce largelibraries. Using the “phage method” (Scott and Smith, 1990, Science249:386-390; Cwirla, et al., 1990, Proc. Natl. Acad. Sci., 87:6378-6382;Devlin et al., 1990, Science, 249:404-406), very large libraries can beconstructed (10⁶-10⁸ chemical entities). A second approach usesprimarily chemical methods, of which the Geysen method (Geysen et al.,1986, Molecular Immunology 23:709-715; Geysen et al. 1987, J.Immunologic Method 102:259-274) and the recent method of Fodor et al.(1991, Science 251, 767-773) are examples. Furka et al. (1988, 14thInternational Congress of Biochemistry, Volume 5, Abstract FR:013;Furka, 1991, Int. J. Peptide Protein Res. 37:487493), Houghton (U.S.Pat. No. 4,631,211, issued December 1986) and Rutter et al. (U.S. Pat.No. 5,010,175, issued Apr. 23, 1991) describe methods to produce amixture of peptides that can be tested as agonists or antagonists.

In another aspect, synthetic libraries (Needels et al., 1993,“Generation and screening of an oligonucleotide encoded syntheticpeptide library,” Proc. Natl. Acad. Sci. USA 90:10700-4; Ohlmeyer etal., 1993, Proc. Natl. Acad. Sci. USA 90:10922-10926; Lam et al.,International Patent Publication No. WO 92/00252; Kocis et al.,International Patent Publication No. WO 9428028, each of which isincorporated herein by reference in its entirety), and the like can beused to screen for choline binding protein ligands according to thepresent invention.

The screening can be performed with recombinant cells that express thecholine binding protein, or alternatively, using purified protein, e.g.,produced recombinantly, as described above. For example, the ability oflabeled, soluble or solubilized choline binding protein that includesthe ligand-binding portion of the molecule, to bind ligand can be usedto screen libraries, as described in the foregoing references.

The following examples are presented in order to more fully illustratethe preferred embodiments of the invention. They should in no way beconstrued, however, as limiting the broad scope of the invention.

EXAMPLES Preliminary Considerations

The present invention identifies a family of CBPs which are surfaceexposed proteins and serve as adhesins critical for the binding ofpneumococcus to eukaryotic cells found at the sites of infection. Theinvention also identifies and characterizes a novel CBP, CbpA, as anadhesin and a determinant of virulence. Furthermore, the presentinvention demonstrates that these proteins are immunogenic and thus mayalso function as protective antigens.

Using choline bound to agarose beads, a family of at least 12 bacterialcholine binding proteins (CBPs), including LytA, was purified from aserotype II, PspA⁻ strain of pneumococcus. Labeling of whole bacteriawith fluorescein isothiocyanate (FITC) suggested that four of these CBPswere surface-exposed while human convalescent sera cross-reacted withfive of the CBPs. It was previously shown that pneumococcus binds totype II lung cells (LC) and endothelial cells (EC) of the peripheralvasculature. Compared to controls, the CBP fraction blocked pneumococcaladherence by 45% to LC and 89% to EC in a dose-dependent manner. The CBPfraction was used to immunize rabbits, and polyclonal antibodies wereused for passive immunization studies. N-terminal and internal aminoacid sequence obtained from three of the CBPs showed no similarity toany known protein sequences. Moreover, the predominant member of thisfamily of proteins, CbpA, is a 112 kDa surface exposed protein thatreacted with human convalescent sera. Sequence analysis of thecorresponding gene showed a unique N-terminal sequence and sixC-terminal choline binding domains in the C-terminal region.

Example 1 Identification of Pneumococcal Choline Binding ProteinsMaterials and Methods

Strains. Bacterial strains used included (1) R6, an unencapsulatedbacterial strain derived from D39 (type 2), unencapsulated, (2) AIIencapsulated, (3) LM91 derived from D39 (encapsulated), which is PspA⁻,and (4) Lyt⁻ derived from D39, unencapsulated.

Cell culture and choline binding protein purification. Cultures of 0.1ml bacteria LM91 (−80° C. stock) were cultivated in 10 ml C+Ysemisynthetic medium for 5 hours at 37° C. This 10 ml culture was thenused to inoculate 400 ml C+Y, and this culture was incubated for 5 hoursat 37° C. in 5% CO₂ to an OD₆₂₀ of 0.6. To this mixture was added 4 mlcholine agarose beads (CAB), and this was incubated 30 minutes at 37° C.in 5% CO₂, with occasional shaking. The bacteria and beads werecentrifuged at 4° C. for 15 minutes at 8000×g, and the pellet wasresuspended in 50 ml rest medium (C+Y). To this 50 ml was added 20 μg/mlLeupeptin, 100 μg/ml PMSF and 250 μl Triton X100 to a finalconcentration of 0.5%. This mixture was rotated for 20-30 minutes untilthe solution became clear, indicating that all bacteria were lysed. TheCAB were then washed on a glass filter with DPBS/1 M NaCl. The CAB werethen incubated with 2 changes of 50 ml DPBS/1 M NaCl, and the beads wereagain washed on a glass filter.

Purification of choline binding proteins. Choline binding proteins(CBPs) were eluted with 3 washes of 5 ml DPBS/10% choline. The elutedCBPs were concentrated on an Ultrafree-20 concentrator (Millipore) to1.5 ml, and then dialyzed against PBS.

Preparation of rabbit anti-CBP antiserum. Choline binding proteinsisolated from the choline affinity column as described above wereconcentrated and used to immunize rabbits in a vaccine to generate ananti-CBP antiserum. New Zealand White rabbits were immunized byintradermal injection in the back with 500 μg purified CBPs in 1 ml of a1:1 emulsion of buffer and Complete Freund's Adjuvant. The rabbits wereboosted with a subcutaneous dorsal injection of 250 μg of purified CBPsin 1 ml of 1:1 buffer to Incomplete Freund's Adjuvant at weeks 3, 6, 9,12, and 15. Rabbits were pre-bled, and test bled approximately 10 daysprior to each boost. Serum obtained at week 16 were used in allexperiments.

The CBPs were examined by SDS-PAGE and Western blot using the followingantibodies: (1) polyclonal anti-pneumococcal serum (rabbit or “ROB”);(2) anti-PspA monoclonal antibody; (3) anti-FITC monoclonal antibody;(4) convalescent human anti-pneumococcal sera and (5) polyclonalanti-CBP antibody. 9 CBPs were identified on SDS-PAGE/Western blot.Results are shown in FIGS. 1 and 2.

Example 2 Characterization of Pneumoccal Choline Binding Proteins

The CBPs isolated by SDS-PAGE were subjected to N-terminal amino acidsequence analysis. The protein fraction obtained from a choline agarosecolumn eluted from 0.5 M choline chloride was applied to an SDSpolyacrylamide gel transferred to a PVDF membrane, and stained forprotein. Individual bands were excised and N-terminal amino acidsequence obtained by Edman degradation. Internal sequence data wasobtained from protease generated peptide fragments. Amino acid sequencedata for 8 of the 9 CBPs were obtained as follows:

CBP112 (SEQ ID NO: 1) XENEGSTQAATSSNMAKTEHRKAAKQVVDE CBP90 (SEQ ID NO:2) AREFSLEKTR CBP84 (SEQ ID NO: 3) XREFSLEKTRNIGIMAHVDAGKT CBP80 (SEQ IDNO: 4) XKXXWQXKQYLKEDGSQAANEXVFDTA CBP78 (SEQ ID NO: 5)QKIIGIDLGTTNSAVAVLEGTESKIIANPE CBP70 (SEQ ID NO: 6) XXXEVAKXSQDTTTASCBP60 (SEQ ID NO: 7) XNERVKIVATLGPAVEGRG CBP50 (SEQ ID NO: 8)XIIXXVYAREVLDSRGNP CBP112-Int1 (SEQ ID NO: 9) EDRRNYHPTNTYK CBP112-Int2(SEQ ID NO: 10) XDDQQAEEDYA

None of the sequences had been previously identified, except that CBP84was 85% identical to Elongation Factor G (EF-G) and CBP50 was similar toenolase.

Example 3 Pneumococcal Choline Binding Proteins Block Adherence

The CBP fraction isolated in Example 1 was used in an adherence assaysto determine its effect on pneumococcal adherence to type II lung cells(LC) and endothelial cells of the vasculature (EC) as described inInternational Patent No. PCT/US95/07209, filed Jun. 6, 1995 by Tuomanenand Cundell, which is specifically incorporated herein by reference inits entirety. The CBP fraction blocked pneumococcal adherence by 45% toLC and 89% to EC in a dose-dependent manner (FIGS. 3 and 4,respectively).

Example 4 The 50 KD Choline Binding Protein Mediates PneumococcalAdhesion

Adherence to extracellular matrix proteins, such as fibronectin (seeFIG. 5), affords pathogens with a means to invade injured epithelia. S.pneumoniae is known to adhere to immobilized fibronectin at a sitewithin the C-terminal heparin binding domain [van der Flier, et. al.Infect. Immun. 63:4317-4322(1995)]. Others have reported that thisregion also binds the leukocyte integrin adhesion receptor α4β1. It hasalso been demonstrated that vascular cell adhesion molecule-1 (VCAM-1)contains sequences homologous to IIICS that are the active binding sitesfor α4β1. This Example evaluates, inter alia, if S. pneumoniae, likeα4β1, cross-recognized fibronectin and VCAM-1.

Adherence of S. pneumoniae to immobilized fibronectin was inhibited 96%by preincubation of the bacteria with recombinant soluble VCAM-1. S.pneumoniae also adhered directly to VCAM-1 coated wells at a density of75±18 bacteria/0.25 mm². This represents 6% of the adherence ofpneumococci to whole fibronectin. S. pneumoniae adherence specificitywas further characterized by evaluating adherence to fragments of the 33kDa heparin binding domain of fibronectin. Various fragments of the 33kDa domain were expressed as GST-fusion proteins. The results are shownin FIG. 6. Bacteria adhered about equally to recombinant fragments33/66, 51, and 15. Further studies using four synthetic peptides basedon the III14 region identified the peptide FN5 (WQPPRARI (SEQ ID NO:11))as able to support adherence of S. pneumoniae (FIG. 7). Antibody to FN5inhibited adherence of pneumococci to both whole fibronectin and VCAM-1(92 and 69% respectively). This Example shows that S. pneumoniae bindsby similar mechanisms to VCAM-1 and fibronectin, which may providepneumococci with access to leukocyte trafficking pathways, promoting theprogression of disease.

One adhesin on the surface of. S. pneumoniae is the 50 kDa cholinebinding protein. Preincubation of bacteria in a 10% choline solution, todisplace surface proteins bound to choline, resulted in a greater than50% decrease in binding of pneumococci to fibronectin (FIG. 8). Bacteriagrown in the presence of ethanolamine instead of choline, so that theydo not display choline binding proteins on their surfaces, failed tofind to fibronectin (>85% inhibition)(data not show). Pneumococcalbinding was decreased by greater than 95% by 1:10 to 1:5000 dilutions ofthe anti-choline antibody, TEPC15 (FIG. 9). Preparations of cholinebinding proteins from S. pneumoniae competitively inhibited theadherence of pneumococci to fibronectin (75-90% inhibition)(data notshown).

To confirm the identity of the protein adhesin on the surface of thepneumococcus, whole cell French Press lysates and preparations ofcholine binding proteins were separated by SDS-PAGE and blotted toImmobilon PVDF membranes. The membranes were then incubated with afibronectin solution for several hours. After washing away unboundprotein, bound fibronectin was detected by chemiluminescence. A band ofapproximately 50 kDa was observed in both the whole cell lysate andcholine binding protein preparations. Amino-terminal protein sequencingof this band identified this protein as a homolog of the glycolyticenzyme, enolase (2-phospho-D-glycerate hydrolyase). In the 18 amino acidstretch analyzed by the BLAST algorithm, a 16 residue region was shownto have 93% identity (15/16) with the enolase from Bacillus subtilis(FIG. 10).

Addition of the glycolytic enzyme enolase (Sigma) to fibronectin coatedwells prior to addition of bacteria resulted in inhibition ofpneumococcal adherence to fibronectin (FIG. 11). Inhibition was dosedependent. Maximum inhibition (98%) was observed with 1000 U/ml enolase.No direct adherence of pneumococcus to enolase coated wells wasobserved.

Enolase has been shown to adhere to plasminogen via its carboxy-terminallysine residue. Adherence of S. pneumoniae to whole fibronectin isL-lysine dependent. Lysine was added to fibronectin coated wells 15 minprior to addition of bacteria. Pneumococcal adherence was inhibited 67%,95%, and 99% by 0.1M, 0.5M, and 1M L-lysine, respectively (FIG. 12). Thelysine analog, 6-amino hexanoic acid, was not able to inhibit adherence.

S. pneumoniae French Press lysates were run over a column of fibronectincoated sepharose beads. Adherent proteins were competitively eluted withL-lysine (0.01-0.5 M) and analyzed by SDS-PAGE. A band of approximately50 kDa was observed in all fractions (FIG. 13). This band comigratedwith commercial enolase (Sigma).

Partial sequence for the enolase gene and the deduced amino acidsequence been obtained (SEQ ID NOS: 10 and 20). Comparison of the DNAand deduced amino acid sequences of pneumococcus and B. subtilis areshown in FIGS. 14 and 15, respectively. There is 74% identity at the DNAlevel, and 72% identity, 85% similarity, at the amino acid level. Morecomplete sequence data for the CBP-50 enolase, lacking only a putative42 base 5′ stretch/14 amino acid N-terminal stretch are shown in SEQ IDNOS:18 and 19, respectively.

In addition to binding to fibronectin and to VCAM, the enolase homologhas two other properties related to virulence. It binds to alpha 1 acidglycoprotein (AGP) which is decorated with carbohydrates that are thekey determinants of binding to activated eukaryotic cells. AGP wasaffixed to a column and a pneumococcal lysate was passed over the columnand the proteins retained on the column were eluted. A 50 kDa proteineluted specifically and upon amino acid N-terminal sequencing was shownto be the enolase homolog. A second property of the enolase homolog isthat it appears to be absent from avirulent pneumococci which have phasevaried to the opaque colonial morphology. When proteins eluted frompneumococci treated with 2% choline are compared between opaque andtransparent strains, the opaque are missing a 112 kDa protein and the 50kD protein. This indicates that lack of virulence is associated withlack of expression of the enolase homolog and therefore enolase homologis a true virulence determinant.

Materials and Methods

Bacterial strains and growth conditions. S. pneumoniae Type 2 (D39) andthe isogenic, unencapsulated derivatives R6x and R6 were used.Pneumococcal strain LM34 deleted for production of pneumococcal surfaceprotein A (pspA−) has been described [McDaniel et al., Infect. Immun.,59:222-9 (1991)]. Bacteria were grown on tryptic soy agar containing 5%sheep blood for 16-18 hours at 37° C. in 5% CO₂ or in a candleextinction jar.

Fibronectin and derived proteolytic fragments. Fibronectin from humanplasma was purchased from Sigma (St. Louis, Mo.) and from Gibco BRL(Grand Island, N.Y.). Fragments designated by molecular size aredepicted schematically in FIG. 5. Proteolytic fragments purchased fromGibco BRL (indicated in parentheses in FIG. 5) included a tryptic 30 kDcollagen binding fragment, an α-chymotryptic 120 kD cell bindingfragment and an α-chymotryptic 40 kD heparin binding fragment.Proteolytic fragments prepared following the method of Vercelotti et al[Vercellotti et al., J. Lab. Clin. Med., 103:34-43 (1984); McCarthy etal., 1986, supra] (indicated in the boxes in FIG. 5) included a 27 kDamino terminal fragment, a 46 kD collagen binding fragment, a 75 kD cellbinding fragment, a 33 kD heparin binding fragment associated with asecond 66 kD fragment (33/66 kD fragment), and a 31 kD carboxy terminalfragment. Recombinant fragments from the 33 kD domain (see FIG. 5) wereprepared as described elsewhere [Huesbsch et al., Cir. Res. 77:43(1995); Verfaille et al., Blood 84:1802 (1994)].

Binding assay to immobilized fibronectin, VCAM and fragments.Fibronectin, its recombinant fragments, or rsVCAM were noncovalentlyimmobilized through passive adsorption to 60 well Terasaki trays(wettable polystyrene [plasma treated], Robbins Scientific, Sunnyvale,Calif.). Briefly, substrates were reconstituted (50 mg/mg) in phosphatebuffered saline (DPBS) and allowed to coat the Teresaki trays for 90 minat 37° C. The polystyrene surface was subsequently blocked with 5%bovine serum albumin (Sigma) for at least 3′ hours at 37° C. Prior touse, the plates were washed five times with DPBS and excess liquid wasremoved from the wells. This procedure was shown to result in thepreferred dense, multilayer packing of adsorbed fibronectin molecules(2.7 μg/cm²) [van der Flier et al., 1995, supra].

Bacteria were labeled with fluorescein isothyocyanate as described[Geelen et al., Infect. Immun., 61:1538-1543 (1993)] and wereresuspended in DPBS supplemented with 0.05% glucose and Ca⁺⁺ and Mg⁺⁺.Bacterial suspensions were brought to an A₆₂₀ of 0.04, previouslyestablished to equal 1×10⁷ cfu/ml of S. pneumoniae. 10 μl of bacteriawere added to each well and incubated statically for 1 hour at 37° C.Unbound bacteria were eliminated by washing five times with DPBS. Boundbacteria were fixed to the surface by incubation with 2.5%glutaraldehyde solution for 3 minutes. Bacteria were counted visuallywith an inverted microscope (Nikon) equipped for fluorescence with an IFDM 510 filter. Binding was expressed as the number of attached bacteriaper 0.25 mm² surface area. Values were corrected for non-specificbinding by subtracting adherence to uncoated wells and are presented asthe mean±standard deviation of at least 3 experiments with 3 to 6wells/plate/experiment. For some experiments, binding was performed inthe presence of rabbit polyclonal anti-pneumococcus R6 antibody or oneof the following monoclonal antibodies: anti-choline (TEPC-15),anti-PspA Xi126 and XiR278 [McDaniel et al., Microb. Pathog., 13:261-9(1992)]; anti-pneumococcal surface adhesin A PsaA [Sampson et al.,Infect. Immun., 62:319-324 (1994)]; anti-VLA-4 (R & D Systems); andanti-VCAM-1 (R & D Systems). Alternatively, potential inhibition ofpneumococcal binding was assessed in the presence of purifiedrecombinant PspA or PsaA.

Example 5 The 112 KD Choline Binding Protin, CbpA Mediates PneumococcalAdhesion to Human Cells

In this Example, the novel 112 kDa CBP is further characterized. Itfunctions as an adhesin, mediating adherence to cytokine activated humancells and participates in pneumococcal colonization of the nasopharynx,and is a determinant of virulence.

Using choline immobilized on a solid matrix, a family of CBPs waspurified from a pspA⁻ strain of pneumococcus. Polyclonal antibody tothese CBPs passively protected mice infected intraperitoneally with alethal challenge of pneumococci. The predominant member of this familyof proteins, CbpA (or CBP-112), is a 112 kDa surface exposed proteinthat reacts with human convalescent sera. Sequence analysis of thecorresponding gene shows a unique N-terminal sequence and a C-terminaldomain comprised of 10 repeated choline binding domains similar to PspA.A cbpA defective mutant shows a greater than 50% reduction in adherenceto cytokine activated human cells and failed to bind to immobilizedsialic acid or laco-N-neotetraose. This mutant also shows a 100-foldreduction in virulence in an animal model for nasopharyngealcolonization. There is no difference between the parent strain and thismutant to an intraperitoneal model for sepsis. This data for CbpAextends the important functions of the CBP family to bacterial adherenceand virulence and represents a new pneumococcal vaccine candidate.

Material and Methods

Strains and media. Serotype 2 S. pneumoniae strain D39, R6x(uncapsulated derivative of D39) strain SIII (capsular serotype 3) (eachobtained from The Rockefeller University collection). Strain P317 is a6B capsular serotype. A lytA deficient strain has been described[Tomasz, 1988]. LM91 was reported previously (Larry McDaniel, Universityof Alabama, Birmingham, Ala.). All strains were plated on tryptic soyagar supplemented with sheep blood (TSAB) to a final concentration of 3%(v/v). Cultures were grown without aeration at 37° C. in 5% CO₂ in aliquid semi-synthetic casein hydrolysate medium supplemented with yeastextract (C+Y medium), as described in Example 1. Pneumococci withintegrated plasmids were grown in the presence of 1 mg ml⁻¹erythromycin. Colony morphology was assessed on tryptic soy agar ontowhich 5000 U of catalase (Worthington Biochemical Co., Freehold, N.J.)were added.

Isolation of CBPs. To prepare the immobilized choline affinity matrix,vinylsulfone-activated agarose beads (1 g; Sigma, St. Louis, Mo.) werewashed twice with 10 ml of distilled water and then rotated overnight atroom temperature in 10 mM choline in sodium carbonate buffer, pH 11.5.The beads were again washed with an additional 10 ml PBS to removeunbound choline.

The choline-agarose beads were added to a 400 ml culture of pneumococci(10⁸ cfu/ml) and incubated for 30 min. The bacteria-bead complex werepelleted by centrifugation at 8000×g for 15 min, and resuspended in 50ml C+Y medium. Bacteria were lysed with Triton X-100 (0.5%; rotation for20 min) in the presence of leupeptin (20 mg ml⁻¹) andphenyl-methyl-sulfonyl floride (100 mg ml⁻¹). The lysate was centrifugedat 1000×g for 5 min to pellet the beads. The beads were then washed withthree volumes (30 ml) of phosphate buffered saline (PBS) adjusted to0.5M NaCl, to remove any non specific bound material. Specific cholinebound material was eluted with PBS adjusted to a final centration of 10%choline (w/v). This eluate was dialyzed against PBS and then passedthrough a Centricon 10 ultrafilter (Amicon Inc. Beverly, Mass.). Theretentate contained the CBPs.

Analytical and Immunological Methods. The CBPs were analyzed by 7.5%sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) andby Western is blotting after electrophoretic transfer to an Immobilon-Ptransfer membrane (Millipore Corporation, Bedford, Mass.). For someexperiments, pneumococci were labelled with fluorescein isothiocyanate(FITC) as described in the adherence assay. Western blot analysis wascarried out with anti-FITC (Moleular Probes), anti-CBP or covalescentantisera each at a dilution of 1:1000. Bound antibody was detected withperoxidase conjugated goat anti-rabbit serum detected with theChemiluminesce Kit from Amersham (Cambridge, Mass.). The convalescentsera was a mixture of sera pooled from five patients collected one monthafter recovery from bacteremia and pneumonia. The capsular serotypes ofthese infecting pneumococci were 3, 7, 14, 22, and 23.

For Western analysis of CBPs in phenotypic variants, opaque andtransparent variants derived from strain R6 were grown to equal densityin C+Y medium (O.D.₆₂₀ of 0.4). The cells were washed in PBS andresuspended in 1/20th the original culture volume in PBS with or without2% choline chloride. The cells were incubated for 15 min. At 4° C. andthe cell and supernatant (eluate) fractions separated. Proteins in theeluate were separated on 10% SDS-PAGE gels, transferred to Immobilon-P,and probed with the antiserum to CBPs.

To obtain primary structural information from individual CBPs,concentrated samples from the choline affinity column were applied toSDS polyacryamide gel and the separated proteins transferred byelectrophoresis to an Immobilon-P^(SQ) transfer membrane. Proteins wereidentified following staining with 0.1% amido black:10% acetic acid:40%methanol. Membranes containing individually stained protein bands wereexcised and submitted to the Rockefeller University Protein SequencingCenter for either N-terminal or internal sequence analysis.

Preparation of rabbit anti-CBP antiserum. Antiserum was generated by HRPInc (Denver, Pa.), according to the methods described in Example 1.

Cloning and sequencing of cbpA. DNA techniques including plasmidpreparations, restriction eridonuclease digests, ligations,transformations into E. coli and gel electrophoresis were according tostandard procedures known in the art.

Initial DNA sequence information for cbpA (SEQ ID NO: 24) was obtainedfrom DNA fragments generated by anchored PCR. These fragments weregenerated using degenerate primers derived from the partial primarystructure of internal fragments of the purified protein (SEQ ID NO: 25)purified by SDS polyacrylamide gel electrophoresis. Further sequenceinformation was obtained from PCR generated fragments produced withtemplates to the end regions of the known fragments and the anchoreduniversal primer. The polymerase chain reaction (PCR) was performedusing the Gene Amp Kit (Perkin Elmer Cetus). DNA sequencing wasperformed on PCR fragments with an ABI373 DNA sequencer using dyelabeled dideoxy terminator chemistry.

A degenerate oligonucleotide corresponding to the amino-terminal aminoacid sequence (SEQ ID NO: 1) of CbpA was prepared(5′GCTCTTNCTCGATGTCTCNGTNGCCAT3′; sense) (SEQ ID NO: 22). The antisenseoligonucleotide (AGCATAGTCTTCTTCGACTTGTTGATCATC) (SEQ ID NO: 23) wasprepared according to the internal amino acid sequence (SEQ ID NO: 10)that was similar to an internal sequence of PspA. LM91 total chromosomalDNA was prepared as described previously and was added (20 ng) to a PCRreaction mixture containing 1 mM each of the sense and antisense primersdescribed above, according to the recommendations provided with AmpliTaqDNA polymerase. Thirty cycles of PCR were performed with oligonucleotidehybridization at 55° C. The DNA band at approximately 600 bp wasisolated following agarose gel electrophoresis with an ultrafree-MCfilter unit (0.45 mm, Millipore Corporation, Bedford, Mass.), digestedwith Sau3A and ligated in the BamHI site of the vector pJDC9. Thismixture was transformed into E. coli DH5α and a single recombinant clonethat contained the vector with the insert was identified.

Further sequence information was obtained from fragments generated by‘anchored’ PCR using primers based on the ends of the known fragmentsand the universal primer anchored to restriction enzyme fragments ofchromosomal DNA. Nucleotide sequencing was performed with an ABI 3373DNA sequencer using dye labeled dideoxy terminator chemistry.

A cbpA deficient mutant was created by insertion duplicationmutagenesis. First, a 643 base pair DNA fragment corresponding tonucleotide 554 through 1196 of cbpA was generated by the PCR. Thisfragment encompasses the coding region for amino acids 155 through 372.A SaulIIA digest of this fragment produced two fragments of 201 and 195base pair which were isolated from an agarose gel, ligated into theBamHI site of pJDC9, and transformed into E. coli (DH5α). A singletransformant that was transformed into the D39 or 6B strains ofpneumococcus and two transformants designated SPRU625 (D39) and SPRU632(6B) were identified that did not express CbpA by Western analysis.Southern analysis confirmed chromosomal integration of the vectordisrupting cbpA in SPRU625 (Data not shown). Chromosomal DNA digestedwith either HindIII or EcoRV was separated by electrophoresis and,transferred bidirectionally to Hybond-N (Amersham). The membranes wereprobed with 643 base pair PCR fragment labeled with horseradishperoxidase (Amersham RPN 3000) as recommended by the manufacturer.

Southern analysis was carried out on electrophoretically separatedchromosomal DNA digested with HindIII and EcoRV, transferredbidirectionally to Hybond-N (Amersham) and probed with the 600 bp PCRfragment labelled with horseradish peroxidase (Amersham RPN 3000) asrecommended by the manufacturer.

Bacterial adhesion assay. The human Type-II lung cell line A549(American Type Culture Collection) was cultured in Nutrient mixture F12.Ham medium (Sigma, St. Louis, Mo.) supplemented with 10% fetal calfserum (Sigma). Human umbilical vein endothelial cells (HUVEC; Clonetics,San Diego, Cailf.) were cultured in medium M199 (Sigma). At confluence,the cells were prepared for subculture with trypsin-0.05% EDTA (Sigma).For adherence assays, cells were transferred to Terasaki 60-well culturedishes (Robbins Scientific, Sunnyvale, Cailf.) and cultured for another24 to 48 hours to form a confluent monolayer. To activate the humancells, some monblayers were incubated with TNFα (10 ng/ml for 2 hours;Boehringer-Manheim) or IL-1β (5 ng/ml for 4 hours; Sigma). Prior to theadherence assay, culture fluid was removed by washing the monolayerstwice with tissue culture medium.

Bacteria at an OD₆₂₀ of 0.4 or 0.6 were washed in 1 ml carbonate buffer(0.05 M sodium carbonate, 0.1 M sodium chloride), resuspended in thesame buffer and labeled with FITC (Sigma; 1 mg ml⁻¹) for 20 min in thedark at room temperature (LO). After 3 washes with carbonate buffer, thebacteria were resuspended in medium M199 without antibiotics and 5×10⁷pneumococci were incubated per well for 30 min at 37° C. in the presenceof 5% CO₂. After the removal of unbound bacteria by washing themonolayers five times with M199, the cells and bacteria were fixed in2.5% glutaraldehyde for 3 min and washed five times with PBS. Adherentpneumococci were counted visually with an inverted microscope(Diaphot-TDM; Nikon Inc., Melville, N.Y.) equipped for epifluorescencewith an IF DM-510 filter and expressed as the number of attachedbacteria per 100 lung cells. Values for 6-9 wells were averaged and eachexperiment was performed 3-6 times.

To test the ability of the mixture of CBPs to affect adherence toeucaryotic cells, the assay was modified such that monolayers wereplated in 96 well dishes (Falcon) coated with 0.2% gelatin and atconfluence were incubated with a range of concentrations of the mixtureof CBPs (1 μg to 1 mg ml⁻¹) for 15 min. After washing, the CBP-treatedmonolayers were challenged with 5×10⁶ pneumococci for 30 min, washed andadherence was quantitated as fluorescence intensity measured in aCytofluor II (Perseptive) with excitation at 485 nm and emission at 530nm.

Adherence to glycoconjugates was assessed by coating Terasaki platesovernight with 100 M of 6′sialyllactose-HSA, lacto-N-neotetraose-HSA,N-acetylglucosamine-β1,4-glucose-HSA orN-acetylglucosamine-β1,3-glucose-HSA (Neose Inc., Horsham, Pa.). Wellswere washed and 1×10⁷ FITC labelled pneumococci were added for 30 min at37° C. Unbound cells were washed away three times with PBS and adherencewas quantitated visually as described above. Each glycoconjugate wastested in 18 wells during three experiments.

Passive protection against systemic challenge. Outbred CF1 mice werehoused under specific pathogen free conditions in accordance withinstitutional and NIH guidelines. Encapsulated pneumococci were grownfor 5 hours in C+Y medium and diluted in PBS. Two groups of ten micereceived an inoculum of 4.5×10⁴ cfu of D39 by injection into theperitoneal cavity. One hour after bacterial challenge, one group of micereceived rabbit-anti-CBP serum intraperitoneally (0.5 ml 1:10 in PBS).Control animals received pre-immune serum. A second experiment wasperformed at a higher challenge dose, with two groups of five mice eachreceiving 8.4×10⁴ cfu of pneumococci. For challenge with SIII, groups offive mice received 200 cfu intraperitoneally of untreated SIII or SIIIpreincubated for 30 min with anti-CBP antiserum. SIII is a highlyvirulent strain with an LD₅₀ of less than 10 bacteria.

Nasopharyngeal challenge. Nasopharyngeal colonization of 1 to 5-day oldSprague-Dawley rats by pneumococci was carried out as describedpreviously using isolates of two capsular serotypes 2 (D39) and 6B(P317). For each experiment, litters were randomized and sorted intogroups ten pups per strain of pneumococci. Each pup received equalintranasal inocula of 10⁴ cfu in 10 ml of PBS of either the parentstrain (D39 or P317) or the isogenic mutant containing a definedmutation in cbpA. As an additional control an isogenic strain (P354)which contains a disruption in the surface IgA1 protease gene, iga, wasused. Colonization was assessed at 24 and 120 hours post-inoculation. Toinsure accurate evaluation of recovered bacteria, the fluid from thenasal washes were diluted in series, plated and colony countsdetermined. Results are expressed as the geometric mean of eachgroup±the standard divination (n=20).

Results

Characterization of CBPs. As in Example 1, a mixture of CBPs wasprepared by incubating a choline affinity matrix with a pspA deficientstrain of bacteria, followed by lysis of cells, treating the beads witha high salt solution to remove non-specifically bound material andeluting the CBPs with a 10% choline solution. The CBP, LytA(muramidase), was tracked through the preparative and analyticalprocedures by western analysis and served as a positive control.Electrophoretic analysis of the CBP preparation showed at lease eightproteins larger than 45 kDa. A protein with an apparent molecular massof 112 kDa was the most abundant. The muramidase, LytA, is present inthe preparation with a molecular mass of 36 kDa, confirming specificityof the technique.

To determine whether this mixture of CBPs contained protective antigens,polyclonal antibody was raised to the CBP mixture and tested for abilityto protect mice passively from intraperitoneal challenge with bacteriaat a concentration 1000-fold greater than the LD₅₀ (FIG. 16A). Nine often animals receiving pre-immune serum died over 3 days in contrast withanimals receiving anti-CBP antiserum. In a second experiment using a2-fold higher inoculum, 100% of the pre-immune treated animals died overone day while all anti-CBP-treated animals survived six days. Theantiserum was then tested for protection against sepsis induced by theheterologous capsule type (Type 3). All control mice receivingintraperitoneal SIII alone or SIII with pre-immune serum were bacteremicat 24 hours with a wide range of bacterial densities from 2×10⁴ to 1×10⁷cfu/ml. In contrast, animals receiving SII with anti-CBP serum oranti-Type III serum showed less than 10⁴ bacteria/ml of blood. Thisdifference was reflected in survival at 36 and 48 hours. Half of controlanimals died by 48 hours while none of the anti-CBP or anti-Type IIIantibody died during the same time interval. Protection was eventuallyovercome and all animals died by 72 hours regardless of treatment.

To determine if the CBP mixture might contain adhesins, the CBP mixturewas tested for its ability to inhibit the adherence of pneumococcus toendothelial and epithelial cells. Pre-incubation of the eukaryotic cellswith the CBPs resulted in a decrease in adherence to epithelial cells byabout 50% and to endothelial cells by greater than 80% (FIG. 17A). Thisphenomenon was dose dependent with half maximal activity requiringapproximately 60 μg/ml of CBP (FIG. 17B).

Based on the indications that the mixture of CBPs contained potentialprotective antigens and adhesive ligands, individual CBPs were studiedfurther. Characteristics were sought which would be important forbioactive CBPs, specifically localization to the pneumococcal surfaceand reactivity with human convalescent antisera and the protectiveanti-CBP serum. Intact pneumococci were labelled with FITC and thensubjected to CBP fractionation. SDS-PAGE and western blot analysis usinganti-FITC antibody showed four proteins labelled with FITC in both wholecell preparations and CBP preparations. This is consistent with accessof this group of CBPs to the pneumococcal extracellular milieu in thenative bacteria. Five CBPs reacted with human convalescent antisera andthe 112 kDa band was prominently labelled in preparations from wholepneumococci. This same pattern occurred using the anti-CBP antiserum,which prominently labelled the 112 kDa band in intact pneumococci andCBP preparations.

As a result of localization of the 112 kDa band to the pneumococcalsurface and its strong reactivity with convalescent antisera andprotective rabbit anti-CBP-sera prepared from whole bacteria or thedescribed CBP fractionation procedure, it was determined that the 112kDa protein is major protein component of the CBP mixture and thusdesignated CbpA.

Cloning and sequence analysis of cbpA. Direct N-terminal amino acidanalysis of CbpA revealed a unique sequence (SEQ ID NO:1) with nosimilarity to proteins in any published data base. In contrast, aninternal sequence (SEQ ID NO:10) was identical to an internal sequenceof the CBP, PspA. Since a pspA deficient strain was used to identifycbpA, this result was not an artifact due to a genetic rearrangement ofpspA but probably represented a gene composed of a unique 5′ codingregion and a 3′ coding region similar to pspA. Using a PCR basedstrategy, cbpA, was cloned and sequenced. Southern analysis using aprobe unique to the 5′ region (see Experimental procedure section)indicated only one copy of cbpA in the LM91 chromosome (data not shown).

Analysis of the derived protein sequence of cbpA brought to lightseveral unique properties. CpbA is a protein of 630 aa with an apparentmolecular mass of 71 kDa. This is different from the calculatedmolecular mass (112 kDa) based on its migration properties by SDS-PAGE.The presence of a standard N-terminal signal sequence implies SecAdependant export. The protein has two distinct domains. The N-terminalregion (1-340) has no obvious similarities to proteins entered intopublished databases. Ganier-Robson analysis predicted six alpha-helicalregions from residues 10-350. Five coiled coil structures greater than50 aa in length in this same region were predicted with the paircoilalgorithm. In contrast, the C-terminal region (341-360) is nearlyidentical (>95%) to PspA. This similarity extends to the correspondingnucleotide sequence. Common features are a proline rich region (340-370)and ten tandem, direct repetitive sequences of 20 aa (381-584) thatrepresent the choline binding motif that is the signature of the cholinebinding proteins.

Phenotypic variation and expression of the choline binding proteins.Since opaque and transparent variants of pneumococci were shown to havedifferences in the expression of the CBP LytA, the expression of theCBPs were compared between opaque and transparent variants of anoncapsulated strain (R6x) with anti-CBP serum. All though all bandswere present in both phenotypes, several bands differed between theopaque and transparent cell types. Transparent organisms hadsignificantly higher amounts of LytA, as previously documented. Inaddition, the transparent variant expressed increased amounts of CbpAand PspA. In contrast, the opaque pneumococci had more PspA. In thesedata minor bands between 70 and 90 kD appeared to be degradationproducts of PspA and CbpA. There was a protein of approximately 50 kDapresent in the transparent organisms regardless of the presence ofcholine suggesting that this was not a CBP. As an added control, theidentity of these CBPs, LytA and PspA, was confirmed by the absence ofthese bands in material obtained from mutants with defects in thecorresponding genes (data not shown).

Functional analysis of CbpA− mutant. To asses bio activities that mightbe affected by CbpA, genetic mutants were constructed in a variety ofassays.

The growth rates of two CbpA⁻ mutants in semisynthetic medium wasequivalent to wild type and the rate of phenotypic variation fromtransparent to opaque colonial variants was not altered from theparental strain (data not shown). Other physiological properties ofpneumococci were also unchanged, including efficiency of DNAtransformation of a streptomycin resistance marker, lysis in stationaryphase, and penicillin-induced lysis (data not shown).

The capsular serotype 2 cbpA deficient mutant was tested for the abilityto adhere to human lung epithelial cells and endothelial cells andglycoconjugates. The mutant adhered to about 80% of parental strainlevels (FIG. 18A, B).

This was consistent with the equivalent ability of the two strains toadhere to immobilized GalNAc-β1,4-Gal and GalNAc-β1,3-Gal, sugars knowto be receptors on these eucaryotic cells (Cundell) (FIG. 18C). Adifference in adherence between the parent and mutant was detected,however, with cytokine activated cells. Adherence of the parental strainto cytokine activated cells increased to ˜135% of resting cell values.In contrast, adherence of the cbpA deficient mutant failed to increasefor either lung or epithelial cells (FIG. 18A). This defect in adherenceto cytokine activated cells was consistent with the absense of adherenceof the mutant to purified glycoconjugates (sialic acid andlacto-N-neotetraose, LnNT) known to be receptors for pneumococci oncytokine activated cells (FIG. 18C).

An infant rat model of pneumoccal carriage was used to determine if theability of CbpA to mediate adherence to human epithelial cellscorrelated with the ability of the pneumococcus to colonize thenasopharynx. In this animal model, the pneumococcus persists on themucosal surface at high levels for many days following a smallintranasal inoculation. In contrast to the controls which were acapsular serotype 2 strain and an isogenic iga deficient mutant (IgA1protease is another surface protein) the cbpA deficient mutant wasgreater than 100 fold less efficient at colonizing the infant ratnasopharynx (FIG. 19). Similar results were obtained comparing acapsular serotype 6B strain (P317) and an isogenic cbpA deficientmutant. The mean bacterial titer in nasal wash at 24 hours for animalschallenged with the parental strain were 3.1×10⁴ cfu/ml (n=9). Incontrast, 6 of 10 pups challenged with the 6B cbpA⁻ deficient strainharbored no detectable bacteria (limit of detection 100 cfu/ml), twopups, 100 cfu/ml and 2 pups, 200 cfu/ml.

In contrast to the results obtained in the nasopharynx, no significantdifferences in virulence were detected in a model for pneumococcalinduced sepsis with a capsular serotype 2, cbpA deficient mutant.

Discussion

To study the unique family of CBP surface proteins, a reverse geneticapproach was developed whereby an immobilized choline matrix was used toselectively capture CBPs from intact pneumococci. The pspA deficientmutant LM91 was chosen as the source of CBPs to avoid purifying the geneproduct. Purification and electrophoretic analysis of the proteinsretained on the choline matrix revealed at least eight CBPs. Severalimportant properties of this mixture were discovered. Antiserum to theCBP mixture protected mice against challenge with the capsular serotype2 strain from which the CBP mixture was derived. In addition, theantiserum decreased the level of bacteremia and prolonged the survivalof mice challenged with a heterologous and highly virulent serotype 3strain. Such cross protection suggests that some CBPs may shareprotective epitopes between serotypes.

In addition to engendering an antiserum with protective activity, theCBP mixture inhibited pneumoccal adherence when applied directly topulmonary epithelial cells and vascular endothelial cells. These cellsare in vitro models for pneumococcal adherence at sites of pneumoccalinfection (NEJM). This suggested that the CBPs bound to receptors on thehuman cells and thereby prevented CBP mediated bacterial attachment. Thedemonstration of a direct CBP human cell interaction that leads to aloss of adherence distinguishes the CBPs from all other pneumoccalproteins that have been identified in genetic studies as affectingbacterial adherence. While loss of these other elements decreases thecapacity of the bacteria to adhere, there is no indication that theyexhibit competitive inhibition of adherence, a key characteristic of astructural adhesin. These adherence and virulence properties of the CBPmixture encouraged further analysis of individual CBPS.

CbpA was chosen for further studies because it was the most abundant CBPin the mixture, it was surface exposed and reacted strongly with bothhuman convalescent antibody and the mouse protective anti-CBP serum,both on whole bacteria and after purification in a CBP preparation.Although the sequence predicts a molecular weight of 71 kDA, the proteinmigrates with an apparent mass of 112 kDa on SDS-PAGE, a discrepancywhich is also characteristic of another CBP, PspA. The choline bindingdomains bf CbpA appears to be identical to those of PspA both at theamino acid an nucleotide level and thus chimeric gene structures forthese elements. The N-terminal domains of the two CBPs are verydifferent in primary sequence, yet they share common structural featuressuch as several large alph-helical regions and five coiled coil regions.This would suggest that CbpA may be a fibrous protein analogous of PspA,tropomyosin, troponin, and the M protein family of streptococcalproteins. It seems reasonable that the N-terminal domain of CbpAcontains one or two lectin domains capable of binding LnNT and sialicacid. Such lectin domains would be expected to remain conserved betweenstrains and serotypes since they must retain the ability to bind thetarget humans glycocojugates receptors in order to retain virulence.This conservation may contribute to cross protection between variousserotypes such as was observed for the anti-CBP antiserum.

CbpA appears to vary in expression coincident with the phase transitionbetween the opaque and transparent variants. Some CBPs, such as PspA,are expressed in higher amounts in opaque variants. CbpA varies in thesame manner as LytA such that transparent variants express increasedamount of this protein. The significance of increased PspA in opaqueorganisms and the function of this CBP is unknown. In contrast theincreased expression of LytA and CbpA by transparent forms correlateswith increased adherence and colonization. A role for LytA incolonization could not be demonstrated. In contrast, the role of CbpA inadherence and colonization was confirmed directly by analysis of thecbpA deficient mutant.

A two step model for pneumoccal adherence has been proposed. Pneumocciinitially target a niche in the host by binding surface glycoconjugates,such as GalNAc-β1,4-Gal and GalNAc-β1,3-Gal, on the surfaces ofeucaryotic cells. A cbpA deficient mutant was unaffected in itsadherence properties to these cells or the correspondingglycoconjugates. A second adhesive step is believed to lead to invasionand is observed upon cytokine activation of the human cell. New sugarspecificities appear on the activated cells resulting in increasedpneumoccal adherence. This second level of adherence is distinguished bythe ability of sialyated glycoconjugates (6′SL) and LnNT to inhibitbacterial attachment. These sugars are not receptor analogs for restingcells. A cbpA deficient mutant lost the ability to bind tocytokine-activated cells or to immobilized 6′SL and LnNT. Together withthe ability of purified CBPs to inhibit pneumococcal attachment, theseresults suggest that CbpA may be a structural adhesin with potentiallectin activity. Cytokine activated cells are suggested to express theplatelet activating factor (PAF) receptor which can bind thephosphorylcholine of the pneumococcal cell wall. The presence of CBPsattached to the phosphorylcholine would presumably maskphosphorylcholine binding to the PAF receptor. The relative number offree versus CBP-bound phosphorylcholine determinants on the pneumoccalsurface is unknown. It appears that both CbpA and phosphorylcholineparticipate in pneumococcal binding to activated human cells.

CbpA appears to represent the first example of a protein adhesin on thepneumococcal surface. It may act as a bridge between cells wallphosphoorylcholine, bound by the C-terminal choline binding repeats, andhuman cell glycoconjugates, presumably via the N-terminal domain. Thebinding capacity is restricted to activated human cells and may,therefore, be important in advancing the course of disease from benigncolonization to invasion. This is also consistent with resultssuggesting CbpA is subject to phase variation and that it is a markerfor the transparent, colonization proficient, phase variants ofpneumococci. Accordingly, CBPs, and CbpA (CBP-112) in particular, areattractive targets for vaccine development and passive immunotherapy.

While the invention has been described and illustrated herein byreferences to various specific material, procedures and examples, it isunderstood that the invention is not restricted to the particularmaterial combinations of material, and procedures selected for thatpurpose. Numerous variations of such details can be implied as will beappreciated by those skilled in the art.

Various references are cited throughout this specification, each ofwhich is incorporated herein by reference in its entirety.

1. A purified antibody to an isolated streptococcal choline bindingprotein which choline binding protein has the following characteristics:a) choline-binding activity; and b) elution from a chromatographiccolumn in the presence of about 10% choline; c) being reactive with serafrom patients infected or recovering from infection with the bacteria;d) being labeled by flourescein isothiocyanate (FITC) without requiringStreptococcal lysis; and e) comprising an amino acid sequence selectedfrom the group consisting of SEQ ID Nos:1,4,6,19 and
 25. 2. The purifiedantibody of claim 1,wherein the purified antibody is a monoclonalantibody.
 3. An immortal cell line that produces a monoclonal antibodyaccording to claim
 2. 4. The antibody of claim 1 or 2 labeled with adetectable label.
 5. The antibody of claim 4 wherein the label isselected from the group consisting of an enzyme, a chemical whichfluoresces, and a radioactive element.
 6. A pharmaceutical compositioncomprising the antibody Of claim 1 or 2 and a pharmaceuticallyacceptable carrier.